Ex vivo proliferation of epithelial cells

ABSTRACT

The technology relates in part to methods and compositions for ex vivo proliferation and expansion of epithelial cells.

RELATED PATENT APPLICATIONS

This patent application is a continuation of international patentapplication no. PCT/US2016/025396 filed on Mar. 31, 2016, entitled EXVIVO PROLIFERATION OF EPITHELIAL CELLS, naming Chengkang Zhang asinventor, and designated by attorney docket no. PPG-2001-PC.International patent application no. PCT/US2016/025396 claims thebenefit of U.S. provisional patent application No. 62/142,851 filed onApr. 3, 2015, entitled EX VIVO PROLIFERATION OF EPITHELIAL CELLS, namingChengkang Zhang as inventor, and designated by attorney docket no.PPG-2001-PV. International patent application no. PCT/US2016/025396 alsoclaims the benefit of U.S. provisional patent application No. 62/217,406filed on Sep. 11, 2015, entitled EX VIVO PROLIFERATION OF EPITHELIALCELLS, naming Chengkang Zhang as inventor, and designated by attorneydocket no. PPG-2001-PV2. International patent application no.PCT/US2016/025396 also claims the benefit of U.S. provisional patentapplication No. 62/294,896 filed on Feb. 12, 2016, entitled EX VIVOPROLIFERATION OF EPITHELIAL CELLS, naming Chengkang Zhang as inventor,and designated by attorney docket no. PPG-2001-PV3. The entire contentof the foregoing applications is incorporated herein by reference,including all text, tables and drawings, for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 17, 2016, isnamed PPG-2001-CTT_SL.txt and is 1,043 bytes in size.

FIELD

The technology relates in part to methods and compositions for ex vivoproliferation and expansion of epithelial cells.

BACKGROUND

Organs such as lung, kidney, liver, pancreas and skin can becharacterized by, among other things, the presence of organ-specificepithelial cells. Epithelial cells may be defined by one or morespecific functions of each such organ. Specific functions may include,for example, gas exchange in the lung, filtration in the kidney,detoxification and conjugation in the liver, insulin production in thepancreatic islet cells or protection against hazardous conditions in theenvironment by the skin. Disease or degeneration of such an organ isoften debilitating or life threatening because degenerated or lost organstructure is not easily replaced, and because the specialized cells ofone organ generally cannot take over the function of another organ.

Certain types of epithelial cells can be difficult to recover and/orregenerate in vivo, and can be challenging to maintain once taken out oftheir context in the body. Certain types of epithelial cells (e.g.,organ-specific epithelial cells harvested from subjects,lineage-committed epithelial cells derived from pluripotent stem cellsand/or differentiated epithelial cells) can be challenging toproliferate and expand in vitro and typically have a very limitedlifespan in culture. To study epithelial cells in vitro or ex vivo, aform of genetic manipulation such as inserting viral or cellularoncogenes, often is required to allow the cells to survive more than afew passages. These genetic manipulations, however, change the geneticbackground and physiology of the cells such that these cells may notresemble or function like normal epithelial cells. Moreover, thesegenetically modified cells would not be candidates for implantation intoan animal.

Methods of culturing and expanding epithelial cells (e.g., cellsharvested from subjects, cells derived from stem cells) for extendedperiods of time, without genetically altering the cells, would be usefulfor a variety of purposes including research applications, personalizedmedicine applications, and transplantation.

SUMMARY

Provided herein in certain aspects are methods for proliferatingdifferentiated epithelial cells ex vivo, which method comprises a)culturing differentiated epithelial cells under serum-free andfeeder-cell free conditions; and b) inhibiting TGF-beta signaling in thedifferentiated epithelial cells during the culturing in (a).

Also provided herein in certain aspects are methods for proliferatingformerly quiescent epithelial cells ex vivo, which method comprises a)culturing formerly quiescent epithelial cells under serum-free andfeeder-cell free conditions; and b) inhibiting TGF-beta signaling in theformerly quiescent epithelial cells during the culturing in (a).

Also provided herein in certain aspects are methods for proliferatinglineage-committed epithelial cells ex vivo, which method comprises a)culturing lineage-committed epithelial cells under serum-free andfeeder-cell free conditions; and b) inhibiting TGF-beta signaling in thelineage-committed epithelial cells during the culturing in (a).

Also provided herein in certain aspects are methods for proliferatingepithelial cells ex vivo, which method comprises a) culturing epithelialcells under feeder-cell free conditions; b) inhibiting TGF-betasignaling in the epithelial cells during the culturing in (a); and c)inhibiting the activity of p21-activated kinase (PAK) in the epithelialcells during the culturing in (a). In some embodiments, the epithelialcells are differentiated epithelial cells, formerly quiescent epithelialcells and/or lineage-committed epithelial cells.

Also provided herein in certain aspects are methods for proliferatingepithelial cells ex vivo, which method comprises a) culturing epithelialcells under serum-free and feeder-cell free conditions; b) inhibitingTGF-beta signaling in the epithelial cells during the culturing in (a);and c) inhibiting the activity of myosin II in the epithelial cellsduring the culturing in (a). In some embodiments, the epithelial cellsare differentiated epithelial cells, formerly quiescent epithelial cellsand/or lineage-committed epithelial cells.

Also provided herein in certain aspects are methods for proliferatingdifferentiated epithelial cells ex vivo, which method comprises a)culturing differentiated epithelial cells under feeder-cell freeconditions; b) activating telomerase reverse transcriptase in thedifferentiated epithelial cells; and c) modulating cytoskeletalstructure in the differentiated epithelial cells.

Also provided herein in certain aspects are methods for proliferatingformerly quiescent epithelial cells ex vivo, which method comprises a)culturing formerly quiescent epithelial cells under feeder-cell freeconditions; b) activating telomerase reverse transcriptase in theformerly quiescent epithelial cells; and c) modulating cytoskeletalstructure in the formerly quiescent epithelial cells.

Also provided herein in certain aspects are methods for proliferatinglineage-committed epithelial cells ex vivo, which method comprises a)culturing lineage-committed epithelial cells under feeder-cell freeconditions; b) activating telomerase reverse transcriptase in thelineage-committed epithelial cells; and c) modulating cytoskeletalstructure in the lineage-committed epithelial cells.

Provided herein in certain aspects is a serum-free cell culture mediumfor proliferating differentiated epithelial cells ex vivo underfeeder-cell free conditions, which serum-free medium comprises one ormore TGF-beta inhibitors (e.g., one or more TGF-beta signalinginhibitors). Also provided herein in certain aspects is a serum-freecell culture medium for proliferating differentiated epithelial cells exvivo under feeder-cell free conditions, which serum-free mediumcomprises a small molecule inhibitor consisting of a TGF-beta inhibitor(e.g., a TGF-beta signaling inhibitor).

Provided herein in certain aspects is a serum-free cell culture mediumfor proliferating formerly quiescent epithelial cells ex vivo underfeeder-cell free conditions, which serum-free medium comprises one ormore TGF-beta inhibitors (e.g., one or more TGF-beta signalinginhibitors). Also provided herein in certain aspects is a serum-freecell culture medium for proliferating formerly quiescent epithelialcells ex vivo under feeder-cell free conditions, which serum-free mediumcomprises a small molecule inhibitor consisting of a TGF-beta inhibitor(e.g., a TGF-beta signaling inhibitor).

Provided herein in certain aspects is a serum-free cell culture mediumfor proliferating lineage-committed epithelial cells ex vivo underfeeder-cell free conditions, which serum-free medium comprises one ormore TGF-beta inhibitors (e.g., one or more TGF-beta signalinginhibitors). Also provided herein in certain aspects is a serum-freecell culture medium for proliferating lineage-committed epithelial cellsex vivo under feeder-cell free conditions, which serum-free mediumcomprises a small molecule inhibitor consisting of a TGF-beta inhibitor(e.g., a TGF-beta signaling inhibitor).

Provided herein in certain aspects is a serum-free cell culture mediumfor proliferating differentiated epithelial cells ex vivo underfeeder-cell free conditions, which serum-free medium comprises one ormore telomerase reverse transcriptase activators and one or morecytoskeletal structure modulators. Also provided herein in certainaspects is a serum-free cell culture medium for proliferatingdifferentiated epithelial cells ex vivo under feeder-cell freeconditions, which serum-free medium comprises small molecules consistingof a telomerase reverse transcriptase activator and a cytoskeletalstructure modulator.

Provided herein in certain aspects is a serum-free cell culture mediumfor proliferating formerly quiescent epithelial cells ex vivo underfeeder-cell free conditions, which serum-free medium comprises one ormore telomerase reverse transcriptase activators and one or morecytoskeletal structure modulators. Also provided herein in certainaspects is a serum-free cell culture medium for proliferating formerlyquiescent epithelial cells ex vivo under feeder-cell free conditions,which serum-free medium comprises small molecules consisting of atelomerase reverse transcriptase activator and a cytoskeletal structuremodulator.

Provided herein in certain aspects is a serum-free cell culture mediumfor proliferating lineage-committed epithelial cells ex vivo underfeeder-cell free conditions, which serum-free medium comprises one ormore telomerase reverse transcriptase activators and one or morecytoskeletal structure modulators. Also provided herein in certainaspects is a serum-free cell culture medium for proliferatinglineage-committed epithelial cells ex vivo under feeder-cell freeconditions, which serum-free medium comprises small molecules consistingof a telomerase reverse transcriptase activator and a cytoskeletalstructure modulator.

Also provided herein in certain aspects is a cell culture medium forproliferating epithelial cells ex vivo under feeder-cell freeconditions, which medium comprises one or more TGF-beta inhibitors(e.g., one or more TGF-beta signaling inhibitors) and one or more PAK1inhibitors. Also provided herein in certain aspects is a cell culturemedium for proliferating epithelial cells ex vivo under feeder-cell freeconditions, which medium comprises small molecules (e.g., small moleculeinhibitors) consisting of a TGF-beta inhibitor (e.g., a TGF-betasignaling inhibitor) and a PAK1 inhibitor. In some embodiments, theepithelial cells are differentiated epithelial cells, formerly quiescentepithelial cells and/or lineage-committed epithelial cells.

Also provided herein in certain aspects is a cell culture medium forproliferating epithelial cells ex vivo under feeder-cell freeconditions, which medium comprises one or more TGF-beta inhibitors(e.g., one or more TGF-beta signaling inhibitors) and one or more myosinII inhibitors. Also provided herein in certain aspects is a cell culturemedium for proliferating epithelial cells ex vivo under feeder-cell freeconditions, which medium comprises small molecules (e.g., small moleculeinhibitors) consisting of a TGF-beta inhibitor (e.g., a TGF-betasignaling inhibitor) and a myosin II inhibitor. In some embodiments, theepithelial cells are differentiated epithelial cells, formerly quiescentepithelial cells and/or lineage-committed epithelial cells.

Provided herein in certain aspects is a population of ex vivoproliferated (e.g., expanded) epithelial cells produced by a methodcomprising a) culturing and proliferating differentiated epithelialcells under serum-free and feeder-cell free conditions; and b)inhibiting TGF-beta signaling in the differentiated epithelial cellsduring the culturing and proliferating in (a).

Provided herein in certain aspects is a population of ex vivoproliferated (e.g., expanded) epithelial cells produced by a methodcomprising a) culturing and proliferating formerly quiescent epithelialcells under serum-free and feeder-cell free conditions; and b)inhibiting TGF-beta signaling in the formerly quiescent epithelial cellsduring the culturing and proliferating in (a).

Provided herein in certain aspects is a population of ex vivoproliferated (e.g., expanded) epithelial cells produced by a methodcomprising a) culturing and proliferating lineage-committed epithelialcells under serum-free and feeder-cell free conditions; and b)inhibiting TGF-beta signaling in the lineage-committed epithelial cellsduring the culturing and proliferating in (a).

Also provided herein in certain aspects is a population of ex vivoproliferated (e.g., expanded) epithelial cells produced by a methodcomprising a) culturing and proliferating epithelial cells underfeeder-cell free conditions; b) inhibiting TGF-beta signaling in theepithelial cells during the culturing and proliferating in (a); and c)inhibiting the activity of p21-activated kinase (PAK) in the epithelialcells during the culturing and proliferating in (a). In someembodiments, the epithelial cells are differentiated epithelial cells,formerly quiescent epithelial cells and/or lineage-committed epithelialcells.

Also provided herein in certain aspects is a population of ex vivoproliferated (e.g., expanded) epithelial cells produced by a methodcomprising a) culturing and proliferating epithelial cells underserum-free and feeder-cell free conditions; b) inhibiting TGF-betasignaling in the epithelial cells during the culturing and proliferatingin (a); and c) inhibiting the activity of myosin II in the epithelialcells during the culturing and proliferating in (a). In someembodiments, the epithelial cells are differentiated epithelial cells,formerly quiescent epithelial cells and/or lineage-committed epithelialcells.

Provided herein in certain aspects is a population of ex vivoproliferated (e.g., expanded) epithelial cells produced by a methodcomprising a) culturing and proliferating differentiated epithelialcells under serum-free and feeder-cell free conditions; b) activatingtelomerase reverse transcriptase in the differentiated epithelial cellsduring the culturing and proliferating in (a); and c) modulatingcytoskeletal structure in the differentiated epithelial cells during theculturing and proliferating in (a).

Provided herein in certain aspects is a population of ex vivoproliferated (e.g., expanded) epithelial cells produced by a methodcomprising a) culturing and proliferating formerly quiescent epithelialcells under serum-free and feeder-cell free conditions; b) activatingtelomerase reverse transcriptase in the formerly quiescent epithelialcells during the culturing and proliferating in (a); and c) modulatingcytoskeletal structure in the formerly quiescent epithelial cells duringthe culturing and proliferating in (a).

Provided herein in certain aspects is a population of ex vivoproliferated (e.g., expanded) epithelial cells produced by a methodcomprising a) culturing and proliferating lineage-committed epithelialcells under serum-free and feeder-cell free conditions; b) activatingtelomerase reverse transcriptase in the lineage-committed epithelialcells during the culturing and proliferating in (a); and c) modulatingcytoskeletal structure in the lineage-committed epithelial cells duringthe culturing and proliferating in (a).

Also provided herein in certain aspects are cell culture compositionscomprising a defined serum-free cell culture medium, a lipids mix, EGF,FGF, albumin, a TGF-beta inhibitor (e.g., a TGF-beta signalinginhibitor), and a Rho kinase inhibitor (e.g., a Rho-associated proteinkinase inhibitor).

Also provided herein in certain aspects are cell culture compositionsconsisting of a defined serum-free cell culture medium, a lipids mix,EGF, FGF, albumin, a TGF-beta inhibitor (e.g., a TGF-beta signalinginhibitor), and a Rho kinase inhibitor (e.g., a Rho-associated proteinkinase inhibitor).

Also provided herein in certain aspects are cell culture compositionscomprising a defined serum-free cell culture medium, a lipids mix, EGF,FGF, albumin, a TGF-beta inhibitor (e.g., a TGF-beta signalinginhibitor), a Rho kinase inhibitor (e.g., a Rho-associated proteinkinase inhibitor), and a beta-adrenergic agonist (e.g., abeta-adrenergic receptor agonist).

Also provided herein in certain aspects are cell culture compositionsconsisting of a defined serum-free cell culture medium, a lipids mix,EGF, FGF, albumin, a TGF-beta inhibitor (e.g., a TGF-beta signalinginhibitor), a Rho kinase inhibitor (e.g., a Rho-associated proteinkinase inhibitor), and a beta-adrenergic agonist (e.g., abeta-adrenergic receptor agonist).

Also provided herein in certain aspects are methods for proliferatingepithelial cells ex vivo, comprising expanding the number of cells in anoriginating epithelial cell population derived from differentiatedtissue under feeder-cell free expansion culture conditions, therebygenerating an expanded epithelial cell population, where the expansionculture conditions comprise an agent that activates telomerase reversetranscriptase in the population and/or inhibits transforming growthfactor beta (TGF-beta) signaling in the population; the originatingepithelial cell population is capable of 25 population doublings or morewhen cultured under the expansion culture conditions; and theoriginating epithelial cell population is capable of no more than 20population doublings when cultured under control culture conditions thatdo not include the agent.

Also provided herein in certain aspects are methods for proliferatingepithelial cells ex vivo, comprising expanding the number of cells in anoriginating epithelial cell population derived from differentiatedtissue under serum-free and feeder-cell free conditions, therebygenerating an expanded epithelial cell population, where the expansionculture conditions comprise an agent that activates telomerase reversetranscriptase in the population and/or inhibits transforming growthfactor beta (TGF-beta) signaling in the population; and the originatingepithelial cell population comprises quiescent and/or formerly quiescentepithelial cells. In certain embodiments, the expansion cultureconditions comprise a second agent that modulates cytoskeletal structurein the population and the control culture conditions do not include thesecond agent.

Also provided herein in certain aspects are methods for proliferatingepithelial cells ex vivo, comprising expanding the number of cells in anoriginating epithelial cell population derived from differentiatedtissue, embryonic stem (ES) cells, or induced pluripotent stem cells(iPSCs) under feeder-cell free expansion culture conditions, therebygenerating an expanded epithelial cell population, where the expansionculture conditions comprise an agent that activates telomerase reversetranscriptase in the population and/or inhibits transforming growthfactor beta (TGF-beta) signaling in the population; the originatingepithelial cell population is capable of 25 population doublings or morewhen cultured under the expansion culture conditions; and theoriginating epithelial cell population is capable of no more than 20population doublings when cultured under control culture conditions thatdo not include the agent.

Also provided herein in certain aspects are methods for proliferatingepithelial cells ex vivo, comprising expanding the number of cells in anoriginating epithelial cell population under serum-free and feeder-cellfree conditions, thereby generating an expanded epithelial cellpopulation, where the expansion culture conditions comprise an agentthat activates telomerase reverse transcriptase in the population and/orinhibits transforming growth factor beta (TGF-beta) signaling in thepopulation; and the originating epithelial cell population comprisesdifferentiated epithelial cells.

Also provided herein in certain aspects are methods for proliferatingepithelial cells ex vivo, comprising expanding the number of cells in anoriginating epithelial cell population under serum-free and feeder-cellfree conditions, thereby generating an expanded epithelial cellpopulation, where the expansion culture conditions comprise an agentthat activates telomerase reverse transcriptase in the population and/orinhibits transforming growth factor beta (TGF-beta) signaling in thepopulation; and the originating epithelial cell population comprisesquiescent and/or formerly quiescent epithelial cells epithelial cells.

Also provided herein in certain aspects are methods for proliferatingepithelial cells ex vivo, comprising expanding the number of cells in anoriginating epithelial cell population serum-free and feeder-cell freeconditions, thereby generating an expanded epithelial cell population,where the expansion culture conditions comprise an agent that activatestelomerase reverse transcriptase in the population and/or inhibitstransforming growth factor beta (TGF-beta) signaling in the population;and the originating epithelial cell population compriseslineage-committed epithelial cells.

Certain embodiments are described further in the following description,examples, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate certain embodiments of the technology and arenot limiting. For clarity and ease of illustration, the drawings are notmade to scale and, in some instances, various aspects may be shownexaggerated or enlarged to facilitate an understanding of particularembodiments.

FIG. 1 shows growth of prostate epithelial cells in regular culturemedium (i.e., control culture conditions). KSFM, Keratinocyte-SFM(Gibco/Thermo Fisher). PrEGM, Prostate Epithelial Cell Growth Medium(Lonza). PD, population doublings.

FIG. 2 shows growth of bronchial epithelial cells in regular culturemedium (i.e., control culture conditions). KSFM, Keratinocyte-SFM(Gibco/Thermo Fisher). PrEGM, Prostate Epithelial Cell Growth Medium(Lonza). PD, population doublings.

FIG. 3A and FIG. 3B show hTERT expression in epithelial cells. HBEC,human bronchial epithelial cells. hTERT, human telomerase reversetranscriptase gene. KSFM, Keratinocyte-SFM (Gibco/Thermo Fisher). LNCaP,human prostate cancer cell line LNCaP Clone FGC (Sigma-Aldrich). PrEC,prostate epithelial cells. PrEGM, Prostate Epithelial Cell Growth Medium(Lonza). n.d., non-detected. p, passage.

FIG. 4 shows a lack of epithelial stem cell marker LGR5 expression inepithelial cells. Polyclonal Lgr5 antibody (LifeSpan, LS-A1235) was usedat 1:50 dilution. Monoclonal TP63 antibody (Santa Cruz, sc-25268) wasused at 1:50 dilution. DAPI, DNA fluorescence stain4′,6-diamidino-2-phenylindole.

FIG. 5A and FIG. 5B show hTERT expression in bronchial epithelial cells.HBEC, human bronchial epithelial cells. hTERT, human telomerase reversetranscriptase gene. KSFM, Keratinocyte-SFM (Gibco/Thermo Fisher). LNCaP,human prostate cancer cell line LNCaP Clone FGC (Sigma-Aldrich). A, ALK5inhibitor A83-01. Y, Rho kinase inhibitor (i.e., Rho-associated proteinkinase inhibitor) Y-27632. n.d., non-detected. p, passage.

FIG. 6A and FIG. 6B show hTERT expression in prostate epithelial cells.PrEC, prostate epithelial cells. hTERT, human telomerase reversetranscriptase gene. KSFM, Keratinocyte-SFM (Gibco/Thermo Fisher). PrEGM,Prostate Epithelial Cell Growth Medium (Lonza). LNCaP, human prostatecancer cell line LNCaP Clone FGC (Sigma-Aldrich). J2, 3T3-J2 feedercells. A, ALK5 inhibitor A83-01. Y, Rho kinase inhibitor (i.e.,Rho-associated protein kinase inhibitor) Y-27632. n.d., non-detected. p,passage.

FIG. 7 shows plating efficiency of prostate epithelial cells on regulartissue culture surface in KSFM in the presence of ALK5 inhibitor A83-01.PrEC, prostate epithelial cells. KSFM, Keratinocyte-SFM (Gibco/ThermoFisher). A, ALK5 inhibitor A83-01.

FIG. 8 shows plating efficiency of bronchial epithelial cells on regulartissue culture surface in KSFM in the presence of ALK5 inhibitor A83-01.HBEC, human bronchial epithelial cells. KSFM, Keratinocyte-SFM(Gibco/Thermo Fisher). A, ALK5 inhibitor A83-01.

FIG. 9 shows plating efficiency of prostate epithelial cells on regulartissue culture surface in KSFM in the presence of ALK5 inhibitor A83-01and Rho kinase inhibitor (i.e., Rho-associated protein kinase inhibitor)Y-27632. PrEC, prostate epithelial cells. KSFM, Keratinocyte-SFM(Gibco/Thermo Fisher). A, ALK5 inhibitor A83-01. Y, Rho kinase inhibitor(i.e., Rho-associated protein kinase inhibitor) Y-27632.

FIG. 10 shows plating efficiency of bronchial epithelial cells onregular tissue culture surface in KSFM in the presence of ALK5 inhibitorA83-01 and Rho kinase inhibitor (i.e., Rho-associated protein kinaseinhibitor) Y-27632. HBEC, human bronchial epithelial cells. KSFM,Keratinocyte-SFM (Gibco/Thermo Fisher). A, ALK5 inhibitor A83-01. Y, Rhokinase inhibitor (i.e., Rho-associated protein kinase inhibitor)Y-27632.

FIG. 11 shows plating efficiency of prostate epithelial cells oncollagen I-coated tissue culture surface in KSFM in the presence of ALK5inhibitor A83-01 and Rho kinase inhibitor (i.e., Rho-associated proteinkinase inhibitor) Y-27632. PrEC, prostate epithelial cells. KSFM,Keratinocyte-SFM (Gibco/Thermo Fisher). A, ALK5 inhibitor A83-01. Y, Rhokinase inhibitor (i.e., Rho-associated protein kinase inhibitor)Y-27632.

FIG. 12 shows plating efficiency of bronchial epithelial cells oncollagen I-coated tissue culture surface in KSFM in the presence of ALK5inhibitor A83-01 and Rho kinase inhibitor (i.e., Rho-associated proteinkinase inhibitor) Y-27632. HBEC, human bronchial epithelial cells. KSFM,Keratinocyte-SFM (Gibco/Thermo Fisher). A, ALK5 inhibitor A83-01. Y, Rhokinase inhibitor (i.e., Rho-associated protein kinase inhibitor)Y-27632.

FIG. 13 shows cell senescence of late passage prostate epithelial cellsand bronchial epithelial cells in KSFM in the presence of Rho kinaseinhibitor (i.e., Rho-associated protein kinase inhibitor) Y-27632. PrEC,prostate epithelial cells. HBEC, human bronchial epithelial cells. KSFM,Keratinocyte-SFM (Gibco/Thermo Fisher). Y, Rho kinase inhibitor (i.e.,Rho-associated protein kinase inhibitor) Y-27632.

FIG. 14 shows growth of prostate epithelial cells. KSFM,Keratinocyte-SFM (Gibco/Thermo Fisher). PrEGM, Prostate Epithelial CellGrowth Medium (Lonza). A, ALK5 inhibitor A83-01. Y, Rho kinase inhibitor(i.e., Rho-associated protein kinase inhibitor) Y-27632.

FIG. 15 shows growth of bronchial epithelial cells. KSFM,Keratinocyte-SFM (Gibco/Thermo Fisher). A, ALK5 inhibitor A83-01. Y, Rhokinase inhibitor (i.e., Rho-associated protein kinase inhibitor)Y-27632.

FIG. 16 shows growth of prostate epithelial cells in the presence ofvarious compounds (5 μM, filled bar; 1 μM, checkered bar; and 0.2 μM,open bar). Control is KSFM (Keratinocyte-SFM (Gibco/Thermo Fisher)) withno compound.

FIG. 17 shows growth of prostate epithelial cells in the presence ofvarious compounds (5 μM, filled bar; 1 μM, checkered bar; and 0.2 μM,open bar). Control is KSFM (Keratinocyte-SFM (Gibco/Thermo Fisher)) withno compound.

FIG. 18 shows growth of human foreskin keratinocytes in the presence ofKSFM and KSFM+A+Y. A, ALK5 inhibitor A83-01. Y, Rho kinase inhibitor(i.e., Rho-associated protein kinase inhibitor) Y-27632. KSFM,Keratinocyte-SFM (Gibco/Thermo Fisher).

FIG. 19 shows growth of prostate epithelial cells in the presence ofKSFM and KSFM+A+Y. A, ALK5 inhibitor A83-01. Y, Rho kinase inhibitor(i.e., Rho-associated protein kinase inhibitor) Y-27632. KSFM,Keratinocyte-SFM (Gibco/Thermo Fisher).

FIG. 20 shows growth of bronchial epithelial cells in the presence ofKSFM and KSFM+A+Y. A, ALK5 inhibitor A83-01. Y, Rho kinase inhibitor(i.e., Rho-associated protein kinase inhibitor) Y-27632. KSFM,Keratinocyte-SFM (Gibco/Thermo Fisher).

FIG. 21 shows karyotyping results at early and late passages of variousepithelial cells cultured in KSFM plus A83-01 and Y-27632. Bottom leftpanel shows representative metaphase chromosome spreads of HFK cells atearly passage (p3). Bottom right panel shows representative metaphasechromosome spreads of HFK cells at late passage (p19). HFK, humanforeskin keratinocytes. HBEC, human bronchial epithelial cells. PrEC,prostate epithelial cells.

FIG. 22 shows average relative length of telomeres in foreskinkeratinocytes cultured in KSFM plus A83-01 and Y-27632 at variouspopulation doublings. The relative length of telomeres is represented asratio (T/S ratio) of telomeric repeats (T) to single copy gene (S) usingquantitative PCR. KSFM, Keratinocyte-SFM (Gibco/Thermo Fisher). A, ALK5inhibitor A83-01. Y, Rho kinase inhibitor (i.e., Rho-associated proteinkinase inhibitor) Y-27632.

FIG. 23 shows growth of transgenic nuclear-localized Red FluorescenceProtein (nRFP)-expressing epithelial cell lines in KSFM with A83-01 andY-27632. HFK, human foreskin keratinocytes. HBEC, human bronchialepithelial cells. PrEC, prostate epithelial cells. nRFP,nuclear-localized Red Fluorescence Protein.

FIG. 24 shows images of transgenic nuclear-localized Red FluorescenceProtein (nRFP)-expressing epithelial cell lines grown in KSFM withA83-01 and Y-27632. HFK, human foreskin keratinocytes. HBEC, humanbronchial epithelial cells. PrEC, prostate epithelial cells. nRFP,nuclear-localized Red Fluorescence Protein.

FIG. 25 shows growth of human foreskin keratinocytes in the presence ofvarious compounds and conditions. HFK, human foreskin keratinocytes. M*,Modified MCDB-153 medium (with 90 μM CaCl2) plus EGF, aFGF, A83-01, andY-27632. EGF, epithelial growth factor. aFGF, acidic fibroblast growthfactor. BSA, Fatty-acid free BSA (Sigma, A8806). rHA, recombinant humanserum albumin expressed in Rice (Sigma, A9731). Lipids mix, ChemicallyDefined Lipid Concentrate (Gibco, 11905-031). AlbuMAX, AlbuMAX® ILipid-Rich BSA (Gibco, 11020-039).

FIG. 26 shows growth of human bronchial epithelial cells in the presenceof various compounds and conditions. HBEC, human bronchial epithelialcells. M*, Modified MCDB-153 medium (with 90 μM CaCl2) plus EGF, aFGF,A83-01, and Y-27632. EGF, epithelial growth factor. aFGF, acidicfibroblast growth factor. BSA, Fatty-acid free BSA (Sigma, A8806). rHA,recombinant human serum albumin expressed in Rice (Sigma, A9731). Lipidsmix, Chemically Defined Lipid Concentrate (Gibco, 11905-031). AlbuMAX,AlbuMAX® I Lipid-Rich BSA (Gibco, 11020-039).

FIG. 27 shows a list of representative genes whose expression levels aredown-regulated or up-regulated in epithelial cells grown in KSFM plusA83-01 and Y-27632 at different passages, compared to epithelial cellsgrown in KSFM at different passages. Gene expression levels in KSFM atp2 is set at 1. Positive numbers indicate the folds of up-regulation,negative numbers indicate the folds of down-regulation. A, ALK5inhibitor A83-01. Y, Rho kinase inhibitor (i.e., Rho-associated proteinkinase inhibitor) Y-27632. KSFM, Keratinocyte-SFM (Gibco/Thermo Fisher).p2, passage 2. p6, passage 6. p13, passage 13. p23, passage 23.

FIG. 28 shows changes in the behavior of epithelial cells cultured inKSFM with A83-01 and Y-27632 (image on the left side) after the additionof 1 mM CaCl2 (image on the right side). A, ALK5 inhibitor A83-01. Y,Rho kinase inhibitor (i.e., Rho-associated protein kinase inhibitor)Y-27632. KSFM, Keratinocyte-SFM (Gibco/Thermo Fisher).

FIG. 29 shows electric resistance across TRANSWELL membrane forbronchial epithelial cells cultured in KSFM with A83-01 and Y-27632 anddifferent calcium concentrations. A, ALK5 inhibitor A83-01. Y, Rhokinase inhibitor (i.e., Rho-associated protein kinase inhibitor)Y-27632. KSFM, Keratinocyte-SFM (Gibco/Thermo Fisher). RA,all-trans-Retinoic acid (Sigma).

FIG. 30 shows growth of human foreskin keratinocytes (HFK) and humanbronchial epithelial cells (HBEC) cultured in KSFM (Keratinocyte-SFM)plus with A83-01 and Y-27632, supplemented with increasingconcentrations of isoproterenol.

FIG. 31 shows differentiation of human bronchial epithelial cells (HBEC)into bronchospheres. The top panel shows cells viewed at lower (4λ)magnification, and the bottom panel shows cells viewed at higher (20λ)magnification. Large bronchospheres with visible lumen are shown in thebottom panel.

FIG. 32A to FIG. 32D show dome-like structures that form in humanbronchial epithelial cell (HBEC) culture (FIG. 32A, FIG. 32B) and humanforeskin keratinocyte (HFK) culture (FIG. 32C, FIG. 32D) in the presenceof high concentration of CaCl₂. FIG. 32A and FIG. 32C are macroscopicimages of whole wells. FIG. 32B and FIG. 32D are microscopic imagesshowing 3D-like structures of miniature domes.

FIG. 33 shows tight junctions that form between human foreskinkeratinocytes (HFK) after induced differentiation in the presence ofhigh concentration of CaCl₂. The presence of intercellular tightjunctions is revealed by immunofluorescence staining of tight junctionprotein ZO-1 using a monoclonal antibody conjugated to Alexa Fluor® 488(ThermoFisher, 339188).

FIG. 34 shows human foreskin keratinocytes (HFK) with increasingtransmembrane electric resistance (TEER) over time inair-liquid-interface differentiation, in the presence of highconcentration of CaCl₂ in KSFM plus A 83-01 and Y-27632. Submerged phaseis indicated by the grayed box.

FIG. 35 shows human foreskin keratinocytes (HFK) form an epidermal-likestructure over time in air-liquid-interface differentiation, in thepresence of high concentration of CaCl₂ in KSFM plus A 83-01 andY-27632. A multi-layer structure is shown, with layers resemblingstratum corneum, stratum granulosm, stratum spinosum, and stratumbasale.

FIG. 36 shows single cell cloning of human foreskin keratinocytes (HFK)in KSFM plus A 83-01, Y-27632 and isoproterenol.

FIG. 37 shows heterogeneity in cellular morphology of human foreskinkeratinocyte (HFK) progeny derived from a single cell.

FIG. 38 shows single cell colony forming efficiency for human foreskinkeratinocytes (HFK) and human bronchial epithelial cells (HBEC) culturedin KSFM plus A 83-01, Y-27632 and isoproterenol.

DETAILED DESCRIPTION

Provided herein are methods and media compositions for proliferatingepithelial cells ex vivo. Epithelial cells (e.g., epithelial cellsproliferated ex vivo) often are co-cultured with a population of feedercells and/or cultured in a conditioned media derived from feeder cells.Reliance on feeder cells, however, can limit where and how epithelialcells are cultured, and can significantly increase the cost of culturingepithelial cells. Use of feeder cells also can be problematic due tocell culture variability caused by undefined biological factors derivedfrom the feeder cells. Variability can lead to inconsistent results,which makes data interpretation challenging due to lack ofreproducibility. Feeder cells also have the potential to introduceunwanted agents (e.g., retroviruses, other pathogens, and immunogenicnonhuman sialic acid such as Neu5Gc) into the cultured epithelial cells.Such culture conditions may not be desirable for certain applicationssuch as, for example, transplantation.

Epithelial cells are typically cultured in medium supplemented withserum (e.g., fetal bovine serum (FBS)). However, in certain instances,serum can be a source of undefined mitogens, and lot-to-lot variationoften is observed. Moreover, serum may be contaminated with infectiousagents such as mycoplasma and viruses. Thus, serum can be an undefinedand variable component of culture medium, and the use of serum canprevent elucidation of defined nutritional and hormonal requirements forcertain cultured cells.

Epithelial cells can be cultured under feeder-cell free conditions withserum-free media that are often supplemented with one or more growthfactors and one or more undefined animal organ extracts. However,epithelial cells cultured under these conditions typically stopproliferating after a few passages, have very limited lifespans, andgenerally cannot be massively expanded ex vivo.

Provided herein are methods and media compositions for proliferating andexpanding epithelial cells ex vivo without the use of feeder cells orfeeder-cell derived conditioned media, and in certain embodiments,without the use of serum. Also provided herein, in certain embodiments,are methods and media compositions for proliferating and expandingepithelial cells ex vivo under defined cell culture conditions. Alsoprovided herein, in certain embodiments, are populations of epithelialcells proliferated and expanded using the methods and compositionsprovided herein.

Epithelial Cells

Provided herein are methods and compositions for proliferating andexpanding epithelial cells ex vivo. An epithelial cell, or epithelium,typically refers to a cell or cells that line hollow organs, as well asthose that make up glands and the outer surface of the body. Epithelialcells can comprise squamous epithelial cells, columnar epithelial cells,adenomatous epithelial cells or transitional epithelial cells.Epithelial cells can be arranged in single layers or can be arranged inmultiple layers, depending on the organ and location.

Epithelial cells described herein can comprise keratinocyte (KE)epithelial cells or non-keratinocyte (NKE) epithelial cells.Keratinocytes form the squamous epithelium that is found at anatomicsites such as the skin, ocular surface, oral mucosa, esophagus andcervix. Keratinocytes terminally differentiate into flat, highlykeratinized, non-viable cells that help protect against the environmentand infection by forming a protective barrier. Examples of keratinocyteepithelial cells include, but are not limited to, dermal keratinocyte,ocular epithelial cells, corneal epithelial cells, oral mucosalepithelial cells, esophagus epithelial cells, and cervix epithelialcells.

NKE cells form the epithelium of the body such as found in the breast,prostate, liver, respiratory tract, retina and gastrointestinal tract.NKE cells typically differentiate into functional, viable cells whichfunction, for example, in absorption and/or secretion. These cellstypically do not form highly keratinized structures characteristic ofsquamous epithelial cells.

NKE cells for use in the methods described herein can be of any type ortissue of origin. Examples of NKE cells include, but are not limited to,prostate cells, mammary cells, hepatocytes, liver epithelial cells,biliary epithelial cells, gall bladder cells, pancreatic islet cells,pancreatic beta cells, pancreatic ductal epithelial cells, pulmonaryepithelial cells, airway epithelial cells, nasal epithelial cells,kidney cells, bladder cells, urethral epithelial cells, stomachepithelial cells, large intestinal epithelial cells, small intestinalepithelial cells, testicular epithelial cells, ovarian epithelial cells,fallopian tube epithelial cells, thyroid cells, parathyroid cells,adrenal cells, thymus cells, pituitary cells, glandular cells, amnioticepithelial cells, retinal pigmented epithelial cells, sweat glandepithelial cells, sebaceous epithelial cells and hair follicle cells. Insome embodiments, NKE cells do not comprise intestinal epithelial cells.

In some embodiments, epithelial cells comprise basal epithelial cells.Basal epithelial cells generally are cells in the deepest layer ofstratified epithelium and multilayered epithelium. Basal epithelialcells may be cells whose nuclei locate close to the basal lamina in apseudostratified epithelium. In some instances, basal epithelial cellsmay divide (e.g., by asymmetric cell division or symmetric celldivision), giving rise to other basal cells and/or other epithelial celltypes (e.g., other cell types in a stratified epithelium, multilayeredepithelium or pseudostratified epithelium). A proportion of basalepithelial cells in some epithelia may have lifelong self-renewcapability and can give rise to other epithelial cell types and basalcells, and sometimes are considered as epithelial stem cells. Theproportion of basal epithelial cells that have lifelong self-renewcapability and are considered as epithelial stem cells varies amongdifferent tissues.

Epithelial cells may be obtained from a subject and/or a cellularsource. Cells obtained from a subject and/or a cellular source may bereferred as an originating epithelial cell population. An originatingepithelial cell population is the input population of epithelial cellsfor expansion by culture conditions described herein (e.g., expansionculture conditions, feeder-cell free expansion conditions). A cellularsource may include a population of embryonic stem (ES) cells, inducedpluripotent stem cells (iPSCs), and the like. In some embodiments, anoriginating epithelial cell population is isolated from an embryo or astem cell culture derived from an embryo. In some embodiments, anoriginating epithelial cell population is isolated from an inducedpluripotent stem cell (iPSC) culture. An originating epithelial cellpopulation can be obtained from a subject in a variety of manners (e.g.,harvested from living tissue, such as a biopsy, plucked hair follicles,body fluids like urine or body-cavity fluids, or isolated fromcirculation). A subject may include any animal, including but notlimited to any mammal, such as mouse, rat, canine, feline, bovine,equine, porcine, non-human primate and human. In certain embodiments, asubject is a human. In some embodiments, a subject is an animal or humanthat has gestated longer than an embryo in a uterine environment andoften is a post-natal human or a post-natal animal (e.g., neonatalhuman, neonatal animal, adult human or adult animal). Typically, asubject is not an embryo. A subject sometimes is a juvenile animal,juvenile human, adult animal or adult human.

In some embodiments, an originating epithelial cell population isisolated from a sample from a subject. A sample can include any specimenthat is isolated or obtained from a subject or part thereof.Non-limiting examples of specimens include fluid or tissue from asubject, including, without limitation, blood or a blood product (e.g.,serum, plasma, or the like), umbilical cord blood, bone marrow,chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid,lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal, ear,arthroscopic), biopsy sample or tissue biopsy, buccal swab, celocentesissample, washings of female reproductive tract, urine, feces, sputum,saliva, nasal mucous, prostate fluid, lavage, semen, lymphatic fluid,bile, tears, sweat, breast milk, breast fluid, hard tissues (e.g.,liver, spleen, kidney, lung, or ovary), the like or combinationsthereof. The term blood encompasses whole blood, blood product or anyfraction of blood, such as serum, plasma, buffy coat, or the like asconventionally defined. Blood plasma refers to the fraction of wholeblood resulting from centrifugation of blood treated withanticoagulants. Blood serum refers to the watery portion of fluidremaining after a blood sample has coagulated. In some embodiments,fetal cells are isolated from a maternal sample (e.g., maternal blood,amniotic fluid).

In some embodiments, epithelial cells may comprise normal, healthy cells(e.g., cells that are not diseased). In some embodiments, epithelialcells may comprise cells that are not genetically altered. In someembodiments, epithelial cells may comprise diseased and/or geneticallyaltered. Diseased epithelial cells may include cells from a subjectcarrying disease-causing mutation(s) (e.g., epithelial cells withgenetic mutation(s) in the CFTR gene). Diseased epithelial cells mayinclude cells from abnormal tissue, such as from a neoplasia, ahyperplasia, a malignant tumor or a benign tumor. In certainembodiments, diseased epithelial cells may include cells that are nottumor cells. In certain embodiments, diseased epithelial cells mayinclude cells isolated from circulation (e.g., circulating tumor cells(CTCs)) of a subject. In certain embodiments, diseased epithelial cellsmay include cells isolated from bodily samples such as, for example,urine, semen, stool (feces), and the like.

In some embodiments, epithelial cells comprise primary cells. In someembodiments, an originating epithelial cell population comprises primarycells. Primary epithelial cells are taken directly from living tissue,such as a biopsy, plucked hair follicles, bodily samples such as a stoolsample, body fluids like urine, semen or body-cavity fluids, or isolatedfrom circulation. In certain instances, primary cells have not beenpassaged. In certain instances, primary cells have been passaged onetime. Primary cells may be isolated from differentiated tissue (e.g.,isolated from epithelium of various organs). Typically, primary cellshave been freshly isolated, for example, through tissue digestion andplated. Primary cells may or may not be frozen and then thawed at alater time. In addition, the tissue from which the primary cells areisolated may or may not have been frozen of preserved in some othermanner immediately prior to processing. Typically, cells are no longerprimary cells after the cells have been passaged more than once. Cellspassaged once or more and immediately frozen after passaging are alsonot considered as primary cells when thawed. In certain embodiments,epithelial cells are initially primary cells and, through use of themethods described herein, become non-primary cells after passaging. Insome embodiments, cells of an originating epithelial cell population aremaintained or proliferated in cell culture after the cells are isolatedfrom differentiated tissue and prior to contacting the originatingepithelial cell population with culture condition described herein(e.g., an expansion culture condition described herein).

In some embodiments, epithelial cells comprise non-primary cells, suchas cells from an established cell line, transformed cells, thawed cellsfrom a previously frozen collection and the like. In certainembodiments, epithelial cells comprise secondary cells. In someembodiments, epithelial cells comprise no cells from an established cellline.

In some embodiments, a culture composition comprises a heterogeneouspopulation of epithelial cells (e.g., comprises a mixture of cell typesand/or differentiation states such as epithelial stem cells, epithelialprogenitors, epithelial precursor cells, lineage-committed epithelialcells, transit-amplifying epithelial cells, differentiating epithelialcells, differentiated epithelial cells, and terminally differentiatedepithelial cells) derived from the same tissue or same tissuecompartment. In some embodiments, a culture composition comprises ahomogenous population of epithelial cells (e.g., does not include amixture of cell types and/or differentiation states) derived from thesame tissue or same tissue compartment. In some embodiments, ahomogeneous population of epithelial cells comprises at least about 90%epithelial cells that are of the same cell type and/or are present atthe same differentiation state. For example, a homogeneous population ofepithelial cells may comprise at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% epithelial cells that are of the samecell type and/or are present at the same differentiation state. In someembodiments, a homogeneous population of epithelial cells comprisesabout 100% epithelial cells that are of the same cell type and/or arepresent at the same differentiation state. In some embodiments,epithelial cells are a homogenous population of basal epithelial cells.In some embodiments, an originating epithelial cell population may beheterogeneous or may be homogeneous. In some embodiments, an expandedepithelial cell population may be heterogeneous or may be homogeneous.

In some embodiments, epithelial cells are characterized by the celltypes and/or differentiation states that are included in, or absentfrom, a population of epithelial cells. In some embodiments, such cellcharacterization may be applicable to an originating epithelial cellpopulation. In some embodiments, such cell characterization may beapplicable to an expanded epithelial cell population. In someembodiments, such cell characterization may be applicable to anoriginating epithelial cell population and an expanded epithelial cellpopulation. In some embodiments, epithelial cells that include aparticular cell type and/or differentiation state comprise at leastabout 50% epithelial cells that are of the particular cell type and/ordifferentiation state. In some embodiments, epithelial cells thatinclude a particular cell type and/or differentiation state comprise atleast about 90% epithelial cells that are of the particular cell typeand/or differentiation state. For example, epithelial cells that includea particular cell type and/or differentiation state may comprise atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%epithelial cells that are of the particular type and/or differentiationstate. Generally, epithelial cells that do not include a particular celltype and/or differentiation state comprise less than about 10% cellsthat are of the particular cell type and/or differentiation state. Forexample, epithelial cells that do not include a particular cell typeand/or differentiation state may comprise less than about 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, or 1% cells that are of the particular cell typeand/or differentiation state.

In certain embodiments, a culture composition or population consistsessentially of a particular type of epithelial cells, referred tohereafter as “the majority cells.” Such culture compositions can includea minor amount of one or more other types of epithelial cells, referredto hereafter as “the minority cells.” The minority cells typically arefrom, or are derived from, the same tissue as the majority cells, andoften are from, or are derived from, the same tissue compartment, as themajority cells. The majority cells can be greater than 50%, greater than60%, greater than 70%, or greater than 80% of the total cells in thecomposition and often are about 90% or more of the total cells in thecomposition, and sometimes are about 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% or more of the total cells in the composition or population.

In some embodiments, a culture composition comprises a heterogeneouspopulation of epithelial cells at different cell cycle phases, such asthe M phase, the G1 phase, the S phase, the G2 phase, and the G0 phasewhich includes senescence and quiescence. In some embodiments, anoriginating epithelial cell population comprises a heterogeneouspopulation of epithelial cells at different cell cycle phases, such asthe M phase, the G1 phase, the S phase, the G2 phase, and the G0 phasewhich includes senescence and quiescence. In some embodiments, anexpanded epithelial cell population comprises a heterogeneous populationof epithelial cells at different cell cycle phases, such as the M phase,the G1 phase, the S phase, the G2 phase, and the G0 phase which includessenescence and quiescence. Epithelial cells at a particular cell cyclephase can make up 1% to 100% of the population.

In some embodiments, epithelial cells comprise cells at one or morestages of differentiation. In some embodiments, such stages ofdifferentiation may be described for an originating epithelial cellpopulation. In some embodiments, such stages of differentiation may bedescribed for an expanded epithelial cell population. In someembodiments, such stages of differentiation may be described for anoriginating epithelial cell population and an expanded epithelial cellpopulation. For example, epithelial cells (or a population of epithelialcells) may comprise epithelial stem cells, epithelial progenitor cells,lineage-restricted epithelial progenitor cells, epithelial precursorcells, lineage-committed epithelial cells, transit-amplifying epithelialcells, proliferating epithelial cells, differentiating epithelial cells,differentiated epithelial cells, quiescent epithelial cells, formerlyquiescent epithelial cells, non-proliferating epithelial cells, andterminally differentiated epithelial cells (e.g., cells that are foundin tissues and organs). Epithelial cells also may compriselineage-committed epithelial cells differentiated and/or derived frompluripotent stem cells (embryonic stem (ES) cells or induced pluripotentstem cells (iPSCs)).

In some embodiments, epithelial cells comprise differentiated epithelialcells. Differentiated epithelial cells may divide, but typically do nothave the capacity for indefinite self-renewal. In some embodiments,differentiated epithelial cells do not acquire the ability todifferentiate into multiple tissue types. Differentiated epithelialcells cultured in conditions described herein generally are moredifferentiated than undifferentiated cells (e.g., stem cells (embryonicor adult), progenitor cells, precursor cells) and are lessdifferentiated than terminally differentiated cells. Differentiatedepithelial cells generally do not include stem cells (embryonic oradult), progenitor cells or precursor cells. In certain instances,differentiated epithelial cells may be referred to as “tissue-specific”and/or “lineage-committed” epithelial cells. In certain instances,differentiated epithelial cells may comprise tissue-specific and/orlineage-committed epithelial cells. In some embodiments, differentiatedepithelial cells comprise quiescent epithelial cells. In someembodiments, differentiated epithelial cells comprise basal epithelialcells.

In some embodiments, epithelial cells comprise quiescent or formerlyquiescent cells. Quiescent cells generally are non-proliferating cells(i.e., non-cycling cells, cells that have withdrawn from the cell cycle,resting cells), and may be characterized as reversibly growth arrested.Under certain conditions, quiescent cells can be induced to proliferate.Quiescent cells may be characterized as existing in the G0 phase of thecell cycle. Quiescent cells that have been induced to proliferate may bereferred to as formerly quiescent cells.

In some embodiments, epithelial cells comprise organ-specific epithelialcells. Organ-specific epithelial cells sometimes are referred to astissue-specific epithelial cells. In some embodiments, organ-specificepithelial cells may differentiate into more specific cell types withina given organ, but generally do not possess or acquire the ability todifferentiate into cells of other types of organs. Organ-specificepithelial cells generally are more differentiated than undifferentiatedcells (e.g., stem cells (embryonic or adult)) and are lessdifferentiated than terminally differentiated cells. Organ-specificepithelial cells generally do not include embryonic stem cells.Organ-specific epithelial cells may or may not include adult stem cells(e.g., adult epithelial stem cells), and organ-specific epithelial cellsmay or may not include progenitor cells or precursor cells.

In some embodiments, epithelial cells comprise lineage-committedepithelial cells. In some embodiments, epithelial cells can compriselineage-committed epithelial cells differentiated from pluripotent stemcells such as embryonic stem (ES) cells and induced pluripotent stemcells (iPSCs). Lineage-committed epithelial cells may divide, buttypically do not have the capacity for indefinite self-renewal. In someembodiments, lineage-committed epithelial cells may differentiate intovarious cell types within a given cell lineage (e.g., respiratory,digestive or integumentary lineages), but generally do not possess oracquire the ability to differentiate into cells of different celllineages (e.g., integumentary lineage-committed epithelial cellsgenerally do not differentiate into blood cells). Lineage-committedepithelial cells generally are more differentiated than undifferentiatedpluripotent stem cells and are less differentiated than terminallydifferentiated cells. Lineage-committed epithelial cells generally donot include pluripotent stem cells (embryonic or induced pluripotent).In some embodiments, lineage-committed epithelial cells includeprogenitor cells or precursor cells. In some embodiments,lineage-committed epithelial cells comprise basal epithelial cells.

In some embodiments, epithelial cells do not include terminallydifferentiated epithelial cells. Terminally differentiated epithelialcells generally do not divide and are committed to a particularfunction. Terminally differentiated epithelial cells generally arecharacterized by definitive withdrawal from the cell cycle and typicallycannot be induced to proliferate. In some embodiments, epithelial cellsdo not include terminally differentiated gastric epithelial cells,intestinal epithelial cells, and/or pancreatic epithelial cells. In someembodiments, epithelial cells do not include post-mitotic cells.Post-mitotic cells generally are incapable of or no longer capable ofcell division. In some embodiments, epithelial cells do not includesenescent cells.

In some embodiments, epithelial cells do not include embryonic stemcells. In some embodiments, epithelial cells are differentiated and/orderived from embryonic stem cells. In some embodiments, epithelial cellsare not derived from embryonic stem cells. Generally, embryonic stemcells are undifferentiated cells that have the capacity to regenerate orself-renew indefinitely. Embryonic stem cells sometimes are consideredpluripotent (i.e., can differentiate into many or all cell types of anadult organism) and sometimes are considered totipotent (i.e., candifferentiate into all cell types, including the placental tissue). Insome embodiments, epithelial cells do not include induced pluripotentstem cells (iPSCs). In some embodiments, epithelial cells aredifferentiated and/or derived from induced pluripotent stem cells(iPSCs). In some embodiments, epithelial cells are not derived frominduced pluripotent stem cells (iPSCs). Generally, induced pluripotentstem cells (iPSCs) are a type of pluripotent stem cell that can begenerated directly from adult cells. In some embodiments, epithelialcells do not include pluripotent cells. In some embodiments, epithelialcells do not include totipotent cells.

In some embodiments, epithelial cells include adult stem cells. Adultstem cells typically are less differentiated than differentiated cells,organ-specific cells or lineage-committed cells and are moredifferentiated than embryonic stem cells. Adult stem cells may bereferred to as stem cells, undifferentiated stem cells, precursor cellsand/or progenitor cells, and are not considered embryonic stem cells asadult stem cells are not isolated from an embryo. Adult epithelial stemcells may be referred to as epithelial stem cells, undifferentiatedepithelial stem cells, epithelial precursor cells and/or epithelialprogenitor cells.

In some embodiments, epithelial cells do not include adult stem cells orcells derived from adult stem cells. In some embodiments, epithelialcells do not include epithelial stem cells or cells derived fromepithelial stem cells. In some embodiments, epithelial cells do notinclude pluripotent epithelial stem cells or cells derived frompluripotent epithelial stem cells. In some embodiments, epithelial cellsdo not include progenitor cells or cells derived from progenitor cells.In some embodiments, epithelial cells do not include precursor cells orcells derived from precursor cells. In some embodiments, epithelialcells do not include continuously proliferating (e.g., continuouslyproliferating in vivo) epithelial stem cells (e.g., intestinal cryptcells; Lgr5+ cells) or cells derived from continuously proliferatingepithelial stem cells. In some embodiments, originating cells, or tissuefrom which originating cells are harvested, do not include continuouslyproliferating epithelial stem cells (e.g., intestinal crypt cells; Lgr5+cells) and methods herein may not include selecting for such cell types.For example, in some embodiments, a method does not include selectingfor continuously proliferating epithelial stem cells and/or selectingfor an in vivo population of continuously proliferating epithelial stemcells (i.e., a population of epithelial stem cells that are continuouslyproliferating in a subject prior to harvest). In some embodiments,epithelial cells do not acquire the ability to form organoids. In someembodiments, epithelial cells are not completely undifferentiated cellsupon initial isolation and plating.

In some embodiments, epithelial cells may be characterized by whetherthe cells possess one or more markers (e.g., cell surface markers,mRNAs, proteins, epigenetic signatures) and/or do not possess measurablelevels of, or possess low levels of, certain markers. In someembodiments, such marker characterization may be applicable to anoriginating epithelial cell population. In some embodiments, such markercharacterization may be applicable to an expanded epithelial cellpopulation. In some embodiments, such marker characterization may beapplicable to an originating epithelial cell population and an expandedepithelial cell population.

In certain instances, level of expression (e.g., mRNA expression) isdetermined for a marker. Levels of mRNA expression may be determinedusing any suitable method for detecting and measuring mRNA expression.For example, expression level may be determined by quantitative reversetranscription PCR according to a Ct_(gene) value, where Ct_(gene) is thenumber of cycles required for a fluorescent signal of a quantitative PCRreaction to cross a defined (e.g., detectable) threshold. Generally,expression of a marker is considered absent if the Ct_(gene) is higherthan 35. The expression level of a marker is considered low if theCt_(gene) is less than 35 and greater than or equal to 30. Theexpression level of a marker is considered medium or moderate if the eneCt_(gene) is less than 29 and greater than or equal to 22. Theexpression level of a marker is considered high if the Ct_(gene) lessthan 22.

In some embodiments, epithelial cells possess markers (e.g., cellsurface markers, mRNAs, proteins, epigenetic signatures) typicallyassociated with a particular cell type and/or differentiation state. Insome embodiments, epithelial cells do not possess markers (e.g., cellsurface markers, mRNAs, proteins, epigenetic signatures) typicallyassociated with a particular cell type and/or differentiation state. Insome embodiments, epithelial cells possess markers (e.g., cell surfacemarkers, mRNAs, proteins, epigenetic signatures) not typicallyassociated with a particular cell type and/or differentiation state. Insome embodiments, epithelial cells possess markers (e.g., cell surfacemarkers, mRNAs, proteins, epigenetic signatures) not typicallyassociated with undifferentiated stem cells. In some embodiments,epithelial cells possess one or more markers (e.g., cell surfacemarkers, mRNAs, proteins, epigenetic signatures) that typically areassociated with basal epithelial cells. In some embodiments, epithelialcells possess one or more markers (e.g., cell surface markers, mRNAs,proteins, epigenetic signatures) that typically are associated withdifferentiated epithelial cells. In some embodiments, epithelial cellspossess one or more markers (e.g., cell surface markers, mRNAs,proteins, epigenetic signatures) that typically are associated withairway epithelial cells and/or keratinocyte cells. Moderate levels tohigh levels of markers may be present in some instances. In someembodiments, epithelial cells do not possess measurable levels of, orpossess low levels of, one or more markers (e.g., cell surface markers,mRNAs, proteins, epigenetic signatures) typically associated withcertain cell types such as, for example, pluripotent stem cells,terminally differentiated epithelial cells, senescent cells, gastricepithelial cells, intestinal epithelial cells, pancreatic epithelialcells, fibroblast cells, and/or intestinal goblet cells. In someembodiments, epithelial cells do not possess measurable levels of, orpossess low levels of, one or more markers (e.g., cell surface markers,mRNAs, proteins, epigenetic signatures) typically associated celladhesion and/or stress response.

In some embodiments, organ-specific epithelial cells do not possessmeasurable levels of, or possess low levels of, one or more markers(e.g., cell surface markers, mRNAs, proteins, epigenetic signatures)typically associated with cell types from other organs. For example,airway epithelial cells may not possess measurable levels of, or possesslow levels of, one or more markers (e.g., cell surface markers, mRNAs,proteins, epigenetic signatures) typically associated with gastricepithelial cells, intestinal epithelial cells, pancreatic epithelialcells, fibroblast cells, and/or intestinal goblet cells.

In some embodiments, epithelial cells possess one or more markers (e.g.,cell surface markers, mRNAs, proteins, epigenetic signatures) thattypically are associated with basal epithelial cells such as, forexample, ITGA6, ITGB4, KRT14, KRT15, KRT5 and TP63. In some embodiments,epithelial cells possess one or more markers (e.g., cell surfacemarkers, mRNAs, proteins, epigenetic signatures) that typically areassociated with differentiated epithelial cells such as, for example,KRT4, KRT6 and KRT8. In some embodiments, epithelial cells possess oneor more markers (e.g., cell surface markers, mRNAs, proteins, epigeneticsignatures) that typically are associated with airway epithelial cellssuch as, for example, HEY2, NGFR and BMP7. In some embodiments,epithelial cells possess one or more markers (e.g., cell surfacemarkers, mRNAs, proteins, epigenetic signatures) that typically areassociated with keratinocyte cells such as, for example, ZFP42. In someembodiments, epithelial cells possess one or more markers (e.g., cellsurface markers, mRNAs, proteins, epigenetic signatures) such as, forexample CDKN2B, CITED2, CREG1, ID1, MAP2K6, IGFBP3 and IGFBP5. Moderatelevels to high levels of such markers may be present in some instances.

In some embodiments, epithelial cells do not possess a measurable levelof, or possess low levels of, one or more markers (e.g., cell surfacemarkers, mRNAs, proteins, epigenetic signatures) typically associatedwith epithelial stem cells such as, for example, LGR5. In someembodiments, epithelial cells do not possess a measurable level of, orpossess low levels of, one or more markers (e.g., cell surface markers,mRNAs, proteins, epigenetic signatures) typically associated withpluripotent stem cells such as, for example, LIN28A, NANOG, POU5F1/OCT4and SOX2. In some embodiments, epithelial cells do not possess ameasurable level of, or possess low levels of, one or more markers(e.g., cell surface markers, mRNAs, proteins, epigenetic signatures)typically associated with terminally differentiated epithelial cellssuch as, for example, CFTR, FOXJ1, IVL, KRT1, KRT10, KRT20, LOR, MUC1,MUC5AC, SCGB1A1, SFTPB and SFTPD. In some embodiments, epithelial cellsdo not possess a measurable level of, or possess low levels of, one ormore markers (e.g., cell surface markers, mRNAs, proteins, epigeneticsignatures) typically associated with cell senescence such as, forexample, AKT1, ATM, CDKN2A, GADD45A, GLB1, PLAU, SERPINE1 and SOD2. Insome embodiments, epithelial cells do not possess a measurable level of,or possess low levels of, one or more markers (e.g., cell surfacemarkers, mRNAs, proteins, epigenetic signatures) typically associatedwith cell adhesion such as, for example, adhesion molecules FN1 andTHBS1. In some embodiments, epithelial cells do not possess a measurablelevel of, or possess low levels of, one or more markers (e.g., cellsurface markers, mRNAs, proteins, epigenetic signatures) typicallyassociated with cell filaments such as, for example, intermediatefilament protein vimentin (VIM). In some embodiments, epithelial cellsdo not possess a measurable level of, or possess low levels of, one ormore markers (e.g., cell surface markers, mRNAs, proteins, epigeneticsignatures) typically associated with gastric epithelial cells,intestinal epithelial cells, or pancreatic epithelial cells such as, forexample, CD34, HNF1A, HNF4A, IHH, KIT, LGR5, PDX1, and PROM1/CD133. Insome embodiments, epithelial cells do not possess a measurable level of,or possess low levels of, one or more markers (e.g., cell surfacemarkers, mRNAs, proteins, epigenetic signatures) typically associatedwith fibroblast cells such as, for example, ZEB1 and ZEB2. In someembodiments, epithelial cells do not possess a measurable level of, orpossess low levels of, one or more markers (e.g., cell surface markers,mRNAs, proteins, epigenetic signatures) typically associated withintestinal goblet cells such as, for example, KRT20.

Cell Culture

Provided herein are methods and compositions for cell culture. Inparticular, provided herein are expansion culture conditions. Cellculture, or culture, typically refers to the maintenance of cells in anartificial, in vitro environment, or the maintenance of cells in anexternal, ex vivo environment (i.e., outside of an organism), and caninclude the cultivation of individual cells and tissues. Certain cellculture systems described herein may be an ex vivo environment and/or anin vitro environment. In some embodiments, primary cells are isolated.In some embodiments, primary cells may be isolated using a single needlebiopsy. In some embodiments, primary cells may be isolated using atissue biopsy. In some embodiments, primary cells may be isolated from aplucked hair. In some embodiments, primary cells may be isolated frombody fluids like urine or body-cavity fluids. In some embodiments,primary cells may be isolated from the circulation of a subject.

After isolation, cellular material may be washed (e.g., with salineand/or a PBS solution). Cellular material may be treated with anenzymatic solution such as, for example, collagenase, dispase and/ortrypsin, to promote dissociation of cells from the tissue matrix.Dispase, for example, may be used to dissociate epithelium fromunderlying tissue. An intact epithelium may then be treated with trypsinor collagenase, for example. Such digestion steps often result in aslurry containing dissociated cells and tissue matrix. The slurry canthen be centrifuged with sufficient force to separate the cells from theremainder of the slurry. A cell pellet may then be removed and washedwith buffer and/or saline and/or cell culture medium. The centrifugingand washing can be repeated any number of times. After a final washing,cells can then be washed with any suitable cell culture medium. Incertain instances, digestion and washing steps may not be performed ifthe cells are sufficiently separated from the underlying tissue uponisolation (e.g., for cells islolated from circulation or using needlebiopsy). In some embodiments, cells such as tumor cells may be isolatedfrom the circulation of a subject. In certain embodiments, tumor cellsmay be isolated according to cell markers specifically expressed oncertain types of tumor cells (see e.g., Lu. J., et al., Intl. J. Cancer,126(3):669-683 (2010) and Yu, M., et al., J. Cell Biol., 192(3): 373-382(2011), which are incorporated by reference). Cells may or may not becounted using an electronic cell counter, such as a Coulter Counter, orthey can be counted manually using a hemocytometer.

Cell seeding densities may be adjusted according to certain desiredculture conditions. For example, an initial seeding density of fromabout 1×10³ to about 1-10×10⁵ cells per cm² may be used. In someembodiments, an initial seeding density of from about 1-10 to about1-10×10⁵ cells per cm² may be used. In certain instances, 1×10⁶ cellsmay be cultured in a 75 cm² culture flask. Cell density may be alteredas needed at any passage.

Cells may be cultivated in a cell incubator at about 37° C. at normalatmospheric pressure. The incubator atmosphere may be humidified and maycontain from about 3-10% carbon dioxide in the air. In some instances,the incubator atmosphere may contain from about 0.1-30% oxygen.Temperature, pressure and carbon dioxide and oxygen concentration may bealtered as needed. Culture medium pH may be in the range of about 7.1 toabout 7.6, or from about 7.1 to about 7.4, or from about 7.1 to about7.3.

Cell culture medium may be replaced every 1-2 days or more or lessfrequently as needed. As the cells approach confluence in the culturevessel, they may be passaged. A cell passage is a splitting or dividingof the cells, and a transferring a portion of the cells into a newculture vessel or culture environment. Cells which are adherent to thecell culture surface may require detachment. Methods of detachingadherent cells from the surface of culture vessels are well known andcan include the use of enzymes such as trypsin.

A single passage refers to a splitting or manual division of the cellsone time, and a transfer of a smaller number of cells into a newcontainer or environment. When passaging, the cells can be split intoany ratio that allows the cells to attach and grow. For example, at asingle passage the cells can be split in a 1:2 ratio, a 1:3 ratio, a 1:4ratio, a 1:5 ratio, and so on. In some embodiments, cells are passagedat least about 1 time to at least about 300 times. For example, cellsmay be passaged at least about 2 times, 5 times, 10 times, 20 times, 30times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100times, 200 times or 300 times. In some embodiments, cells are passagedat least about 15 times. In some embodiments, cells are passaged atleast about 25 times.

Cell growth generally refers to cell division, such that one mother celldivides into two daughter cells. Cell growth may be referred to as cellexpansion. Cell growth herein generally does not refer to an increase inthe actual size (e.g., diameter, volume) of the cells. Stimulation ofcell growth can be assessed by plotting cell populations (e.g., cellpopulation doublings) overtime. A cell population with a steeper growthcurve generally is considered as growing faster than a cell populationwith a less steep curve. Growth curves can be compared for varioustreatments between the same cell types, or growth curves can be comparedfor different cell types with the same conditions, for example.

Expanding a population of cells may be expressed as population doubling.A cell population doubling occurs when the cells in culture divide sothat the number of cells is doubled. In some instances, cells arecounted to determine if a population of cells has doubled, tripled ormultiplied by some other factor. The number of population doublings maynot be equivalent to the number of times a cell culture is passaged. Forexample, passaging the cells and splitting them in a 1:3 ratio forfurther culturing may not be equivalent to a tripled cell population. Aformula that may be used for the calculation of population doublings(PD) is presented in Equation A:

n=3.32*(log Y−log I)+X  Equation A

where n=the final PD number of the cell culture when it is harvested orpassaged, Y=the cell yield at the time of harvesting or passaging, I=thecell number used as inoculum to begin that cell culture, and X=the PDnumber of the originating cell culture that is used to initiate thesubculture.

A population of cells may double a certain number of times over acertain period of time. In some embodiments, a population of cells iscapable of doubling, or doubles, at least about 1 time to at least about500 times over a certain period of time. For example, a population ofcells may be capable of doubling, or double, at least about 2 times, 5times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70times, 80 times, 90 times, 100 times, 110 times, 120 times, 130 times,140 times, 150 times, 160 times, 170 times, 180 times, 190 times, 200times, 250 times, 300 times, 350 times, 400 times, 450 times or 500times. In some embodiments, the cell population is capable of doubling,or doubles, at least 20 times. In some embodiments, the cell populationis capable of doubling, or doubles, at least 50 times. In someembodiments, the cell population is capable of doubling, or doubles, atleast 60 times. In some embodiments, the cell population is capable ofdoubling, or doubles, at least 70 times. In some embodiments, the cellpopulation is capable of doubling, or doubles, at least 80 times. Insome embodiments, the cell population is capable of doubling, ordoubles, at least 90 times. In some embodiments, the cell population iscapable of doubling, or doubles, at least 100 times. In someembodiments, the cell population is capable of doubling, or doubles, atleast 120 times. In some embodiments, the cell population is capable ofdoubling, or doubles, at least 150 times. In some embodiments, the cellpopulation is capable of doubling, or doubles, at least 200 times. Insome embodiments, a population of cells doubles, or is capable ofdoubling, a certain number of times over a period of about 1 day toabout 500 days. For example, a population of cells may double, or iscapable of doubling, a certain number of times over a period of about 2days, 5 days, 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70days, 80 days, 90 days, 100 days, 110 days, 120 days, 130 days, 140days, 150 days, 160 days, 170 days, 180 days, 190 days, 200 days, 250days, 300 days, 350 days, 400 days, 450 days or 500 days. In someembodiments, a population of cells doubles, or is capable of doubling, acertain number of times over a period of about 50 days. In someembodiments, a population of cells doubles, or is capable of doubling, acertain number of times over a period of about 100 days. In someembodiments, a population of cells doubles, or is capable of doubling, acertain number of times over a period of about 150 days. In someembodiments, a population of cells doubles, or is capable of doubling, acertain number of times over a period of about 200 days.

In some embodiments, a method herein comprises expanding a population ofcells. Expanding a population of cells may be referred to asproliferating a population of cells. Expanding a population of cells maybe expressed as fold increase in cell numbers. A formula that may beused for the calculation of fold increase as a function of populationdoublings is presented in Equation B:

F=2^(n)  Equation B

where F=the fold increase in cell numbers after n population doublings.For example, after one (1) population doubling, the number of cellsincreases by 2 fold, and after two (2) population doublings, the numberof cells increases by 4 (2²=4) fold, and after three (3) populationdoublings, the number of cells increases by 8 (2³=8) fold, and so on.Hence, after twenty (20) population doublings, the number of cellsincreases by more than one million fold (2²⁰=1,048,576), and afterthirty (30) population doublings, the number of cells increases by morethan one billion fold (2³⁰=1,073,741,824), and after forty (40)population doublings, the number of cells increases by more than onetrillion fold (2⁴⁰=1,099,511,627,776), and so on. In some embodiments, apopulation of cells is expanded, or is capable of being expanded, atleast about 2-fold to at least about a trillion-fold. For example, apopulation of cells may be expanded at least about 5-fold, 10-fold,15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1,000-fold,10,000-fold, 100,000-fold, 1 million-fold, 1 billion-fold, or 1trillion-fold. A particular fold expansion may occur over a certainperiod of time in culture such as, for example, 2 days, 3 days, 4 days,5 days, 10 days, 20 days, 30 days, 40 days, 50 days, 100 days or more.

Cells may be continuously proliferated or continuously cultured.Continuous proliferation or continuous culture refers to a continuousdividing of cells, reaching or approaching confluence in the cellculture container such that the cells require passaging and addition offresh medium to maintain their health. Continuously proliferated cellsor continuously cultured cells may possess features that are similar to,or the same as, immortalized cells. In some embodiments, cells continueto grow and divide for at least about 5 passages to at least about 300passages. For example, cells may continue to grow and divide for atleast about 10 passages, 20 passages, 30 passages, 40 passages, 50passages, 60 passages, 70 passages, 80 passages, 90 passages, 100passages, 200 passages or 300 passages.

In some embodiments, epithelial cells are a heterogeneous population ofepithelial cells upon initial collection and plating and become ahomogenous population of epithelial cells after one or more passages.For example, a heterogeneous population of epithelial cells may become ahomogeneous population of epithelial cells after 2 passages, after 3passages, after 4 passages, after 5 passages, after 10 passages, after20 passages, after 30 passages, after 40 passages, after 50 passages, orafter 100 or more passages.

In some embodiments, epithelial cells are characterized by the celltypes and/or differentiation states that are included in, or absentfrom, a population of epithelial cells at initial collection andplating. In some embodiments, epithelial cells are characterized by thecell types and/or differentiation states that are included in, or absentfrom, a population of epithelial cells after one or more passages. Forexample, epithelial cells may be characterized by the cell types and/ordifferentiation states that are included in, or absent from, apopulation of epithelial cells after 2 passages, after 3 passages, after4 passages, after 5 passages, after 10 passages, after 20 passages,after 30 passages, after 40 passages, after 50 passages, or after 100 ormore passages. In some embodiments, epithelial cells are characterizedby the cell types and/or differentiation states that are included in anoriginating epithelial cell population. In some embodiments, epithelialcells are characterized by the cell types and/or differentiation statesthat are included in an expanded epithelial cell population.

In some embodiments, cells do not undergo differentiation duringexpansion, continuous proliferation or continuous culture. For example,cells may not differentiate into terminally differentiated cells orother cell types during expansion, continuous proliferation orcontinuous culture. In some embodiments, cells of a particular organ orlineage do not differentiate into cells of a different organ or lineage.For example, airway epithelial cells may not differentiate intofibroblast cells, intestinal epithelial cells, intestinal goblet cells,gastric epithelial cells, or pancreatic epithelial cells duringexpansion, continuous proliferation or continuous culture. In someembodiments, cells undergo some degree of differentiation duringexpansion, continuous proliferation or continuous culture. For example,lineage-committed epithelial cells may differentiate into cell typeswithin a given lineage and/or organ-specific epithelial cells maydifferentiate into other cell types within a given organ duringexpansion, continuous proliferation or continuous culture.

In some embodiments, a certain proportion of the epithelial cells may beat G0 resting phase where the cells have exited cell cycle and havestopped dividing, which includes both quiescence and senescence states.A certain proportion of the epithelial cells may be at G1 phase, inwhich the cells increase in size and get ready for DNA synthesis. Acertain proportion of the epithelial cells may be at S phase, in whichDNA replication occurs. A certain proportion of the epithelial cells maybe at G2 phase, in which the cells continue to grow and get ready toenter the M (mitosis) phase and divide. A certain proportion of theepithelial cells may be at M (mitosis) phase and complete cell division.

In some embodiments, cells are characterized by telomere length. In someembodiments, cells in an originating epithelial cell population arecharacterized by telomere length. In some embodiments, cells in anexpanded epithelial cell population are characterized by telomerelength. Typically, telomere length shortens as cells divide. A cell maynormally stop dividing when the average length of telomeres is reducedto a certain length, for example, 4 kb. In some embodiments, averagetelomere length of cells cultured in media and/or culture conditionsdescribed herein may be reduced to a length of less than about 10 kb,and the cells can continue to divide. For example, average telomerelength of cells cultured in media and/or culture conditions describedherein may be reduced to a length of less than about 9 kb, 8 kb, 7 kb, 6kb, 5 kb, 4 kb, 3 kb, 2 kb, or 1 kb, and the cells can continue todivide. Average telomere length sometimes is expressed as a meantelomere length or median telomere length. Average telomere length maybe determined using any suitable method for determining telomere length,and may vary according to cell type. In some embodiments, averagetelomere length is determined as relative abundance of telomeric repeatsto that of a single copy gene.

In some embodiments, cells are expanded, continuously proliferated orcontinuously cultured for a certain number of passages without alteringcellular karyotype. For example, an alteration in cellular karyotype mayinclude duplication or deletion of chromosomes or portions thereofand/or translocation of a portion of one chromosome to another.Karyotype may be assayed for a population of cells after a certainnumber of passages which may be compared to a population of cells of thesame origin prior to passaging. In some embodiments, cells have anunaltered karyotype after at least about 5 passages to at least about300 passages. For example, cells may have an unaltered karyotype afterat least about 10 passages, 20 passages, 30 passages, 40 passages, 50passages, 60 passages, 70 passages, 80 passages, 90 passages, 100passages, 200 passages or 300 passages. In certain instances, cells thathave an unaltered karyotype after a certain number of passages may bereferred to as conditionally immortalized cells.

In some embodiments, methods herein comprise use of an extracellularmatrix (ECM). In some embodiments, methods herein do not comprise use ofan extracellular matrix. ECM may contain certain polysaccharides, water,elastin, and certain glycoproteins such as, for example, collagen,entactin (nidogen), fibronectin, and laminin. ECM may be generated byculturing ECM-producing cells, and optionally removing these cells,prior to the plating of epithelial cells. Examples of ECM-producingcells include chondrocytes, which produce collagen and proteoglycans;fibroblast cells, which produce type IV collagen, laminin, interstitialprocollagens and fibronectin; and colonic myofibroblasts, which producecollagens (type I, III, and V), chondroitin sulfate proteoglycan,hyaluronic acid, fibronectin, and tenascin-C. ECM also may becommercially provided. Examples of commercially available extracellularmatrices include extracellular matrix proteins (Invitrogen), basementmembrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcomacells (e.g., Matrigel™ (BD Biosciences)), and synthetic extracellularmatrix materials, such as ProNectin (Sigma Z378666). Mixtures ofextracellular matrix materials may be used in certain instances.Extracellular matrices may be homogeneous (comprise essentially a singlecomponent) or heterogeneous (comprise a plurality of components).Heterogeneous extracellular matrices generally comprise a mixture of ECMcomponents including, for example, a plurality of glycoproteins andgrowth factors. Example heterogeneous extracellular matrices includebasement membrane preparations from Engelbreth-Holm-Swarm (EHS) mousesarcoma cells (e.g., Matrigel™). In some embodiments, methods herein donot comprise use of a heterogeneous extracellular matrix. Extracellularmatrices may be defined (all or substantially all components and amountsthereof are known) or undefined (all or substantially all components andamounts thereof are not known). Example undefined extracellular matricesinclude basement membrane preparations from Engelbreth-Holm-Swarm (EHS)mouse sarcoma cells (e.g., Matrigel™). In some embodiments, methodsherein do not comprise use of an undefined extracellular matrix.

In some embodiments, cells are cultured in a container comprising acoating. For example, cells may be plated onto the surface of culturevessels containing one or more attachment factors. In some embodiments,cells are plated onto the surface of culture vessels without attachmentfactors. In embodiments where attachment factors are used, a culturecontainer can be precoated with a natural, recombinant or syntheticattachment factor or factors or peptide fragments thereof, such as butnot limited to collagen, fibronectin and natural or synthetic fragmentsthereof. In some embodiments, a culture vessel is precoated withcollagen. In some embodiments, a culture vessel is precoated with abasement membrane matrix. In some embodiments, a culture vessel isprecoated with a homogeneous and/or defined extracellular matrix.

The cells may maintain one or more functional characteristics throughoutthe culturing process. In some embodiments, a functional characteristicmay be a native functional characteristic. Native functionalcharacteristics generally include traits possessed by a given cell typewhile in its natural environment (e.g., a cell within the body of asubject before being extracted for cell culture). Examples of nativefunctional characteristics include gas exchange capabilities in airwayepithelial cells, detoxification capabilities in liver epithelial cells,filtration capabilities in kidney epithelial cells, and insulinproduction and/or glucose responsiveness in pancreatic islet cells. Insome embodiments, cells do not maintain one or more functionalcharacteristics throughout the culturing process.

A characteristic of cells in culture sometimes is determined for anentire population of cells in culture. For example, a characteristicsuch as average telomere length, doubling time, growth rate, divisionrate, gene level or marker level, for example, is determined for thepopulation of cells in culture. A characteristic often is representativeof cells in the population, and the characteristic may vary forparticular cells in the culture. For example, where a population ofcells in a culture exhibits an average telomere length of 4 kb, aportion of cells in the population can have a telomere length of 4 kb, aportion of cells can have a telomere length greater than 4 kb and aportion of cells can have a telomere length less than 4 kb. In anotherexample, where a population of cells is characterized as expressing ahigh level of a particular gene or marker, all cells in the populationexpress the particular gene or marker at a high level in someembodiments, and in certain embodiments, a portion of cells in thepopulation (e.g., at least 75% of cells) express the particular gene ormarker at a high level and a smaller portion of the cells express theparticular gene at a moderate level, low level or undetectable level. Inanother example, where a population of cells is characterized as notexpressing, or expressing a low level of a particular gene or marker, nocells in the population express the particular gene or marker at adetectable level in some embodiments, and in certain embodiments, aportion of cells in the population (e.g., less than 10% of cells)express the particular gene or marker at a detectable level.

A characteristic of cells in culture (e.g., population doublings, markerexpression) sometimes is compared to the same characteristic observedfor cells cultured in control culture conditions. Often, when comparinga characteristic observed for cells cultured in control cultureconditions, an equal or substantially equal amount of cells from thesame source is added to certain culture conditions and to controlculture conditions. Control culture conditions may include the same basemedium (e.g., a serum-free base medium) and additional components minusone or more agents (e.g., one or more of a TGF-beta inhibitor (e.g., oneor more TGF-beta signaling inhibitors), a ROCK inhibitor, a myosin IIinhibitor, a PAK inhibitor). In some embodiments, cell cultureconditions consist essentially of certain components necessary toachieve one or more characteristics of cells in culture (e.g.,population doublings, marker expression) compared to the samecharacteristic(s) observed for cells cultured in control cultureconditions. When a cell culture condition consists essentially ofcertain components, additional components or features may be includedthat do not have a significant effect on the one or more characteristicsof cells in culture (e.g., population doublings, marker expression) whencompared to control culture conditions. Such additional components orfeatures may be referred to as non-essential components and may includetypical cell culture components such as salts, vitamins, amino acids,certain growth factors, fatty acids, and the like.

Feeder Cells

Cells may be cultured with or without feeder cells. Generally, feedercells are cells co-cultured with other cell types for certain cellculture systems. Feeder cells typically are nonproliferating cells andsometimes are treated to inhibit proliferation, and often are maintainedin a live, metabolically active state. For example, feeder cells can beirradiated with gamma irradiation and/or treated with mitomycin C, whichcan arrest cell division while maintaining the feeder cells in ametabolically active state.

Feeder cells can be from any mammal and the animal source of the feedercells need not be the same animal source as the cells being cultured.For example feeder cells may be, but are not limited to mouse, rat,canine, feline, bovine, equine, porcine, non-human primate and humanfeeder cells. Types of feeder cells may include spleenocytes,macrophages, thymocytes and/or fibroblasts. Types of feeder cells may bethe same cell type which they support. Types of feeder cells may not bethe same cell type which they support. J2 cells are used as feeder cellsfor certain cell culture systems, and are a subclone of mousefibroblasts derived from the established Swiss 3T3 cell line.

In some embodiments, cells are cultured in the absence of feeder cells.In some embodiments, cells are not cultured in media conditioned byfeeder cells (i.e., not cultured in a conditioned medium). In someembodiments, cells are not cultured in the presence of fractionatedfeeder cells, or particulate and/or soluble fractions of feeder cells.Any one or all of the above culture conditions (i.e., cultured in theabsence of feeder cells; not cultured in a conditioned medium; notcultured in the presence of fractionated feeder cells, or particulateand/or soluble fractions of feeder cells) may be referred to asfeeder-cell free conditions or feeder-free conditions. Expansion cultureconditions provided herein typically are feeder-cell free cultureconditions.

Media and Cell Culture Compositions

Cells typically are cultured in the presence of a cell culture medium.Expansion culture conditions provided herein typically comprise a cellculture medium. A cell culture medium may include any type of mediumsuch as, for example, a serum-free medium; a serum-containing medium; areduced-serum medium; a protein-free medium; a chemically definedmedium; a protein-free, chemically defined medium; a peptide-free,protein-free, chemically defined medium; an animal protein-free medium;a xeno-free medium. A cell culture medium typically is an aqueous-basedmedium and can include any of the commercially available and/orclassical media such as, for example, Dulbecco's Modified EssentialMedium (DMEM), Knockout-DMEM (KODMEM), Ham's F12 medium, DMEM/Ham's F12,Advanced DMEM/Ham's F12, Ham's F-10 medium, RPMI 1640, Eagle's BasalMedium (EBM), Eagle's Minimum Essential Medium (MEM), Glasgow MinimalEssential Medium (G-MEM), Medium 199, Keratinocyte-SFM (KSFM;Gibco/Thermo-Fisher), prostate epithelial growth medium (PrEGM; Lonza),CHO cell culture media, PER.C6 media, 293 media, hybridoma media, andthe like and combinations thereof.

In some embodiments, a cell culture medium is a serum-containing medium.Serum may include, for example, fetal bovine serum (FBS), fetal calfserum, goat serum or human serum. Generally, serum is present at betweenabout 1% to about 30% by volume of the medium. In some instances, serumis present at between about 0.1% to about 30% by volume of the medium.In some embodiments, a medium contains a serum replacement.

In some embodiments, a cell culture medium is a serum-free medium. Aserum-free medium generally does not contain any animal serum (e.g.fetal bovine serum (FBS), fetal calf serum, goat serum or human serum),but may contain certain animal-derived products such as serum albumin(e.g., purified from blood), growth factors, hormones, carrier proteins,hydrolysates, and/or attachment factors. In some embodiments, aserum-free cell culture medium comprises Keratinocyte-SFM (KSFM;Gibco/Thermo-Fisher). KSFM may include insulin, transferrin,hydrocortisone, Triiodothyronine (T3). A representative formulation ofKSFM basal medium is described, for example, in U.S. Pat. No. 6,692,961.

In some embodiments, a cell culture medium is a defined serum-freemedium. Defined serum-free media, sometimes referred to aschemically-defined serum-free media, generally include identifiedcomponents present in known concentrations, and generally do not includeundefined components such as animal organ extracts (e.g., pituitaryextract) or other undefined animal-derived products (e.g., unquantifiedamount of serum albumin (e.g., purified from blood), growth factors,hormones, carrier proteins, hydrolysates, and/or attachment factors).Defined media may include a basal media such as, for example, DMEM, F12,or RPMI 1640, containing one or more of amino acids, vitamins, inorganicacids, inorganic salts, alkali silicates, purines, pyrimidines,polyamines, alpha-keto acids, organosulphur compounds, buffers (e.g.,HEPES), antioxidants and energy sources (e.g., glucose); and may besupplemented with one or more of recombinant albumin, recombinant growthfactors, chemically defined lipids, recombinant insulin and/or zinc,recombinant transferrin or iron, selenium and an antioxidant thiol(e.g., 2-mercaptoethanol or 1-thioglycerol). Recombinant albumin and/orgrowth factors may be derived, for example, from non-animal sources suchas rice or E. coli, and in certain instances synthetic chemicals areadded to defined media such as a polymer polyvinyl alcohol which canreproduce some of the functions of bovine serum albumin (BSA)/humanserum albumin (HSA). In some embodiments, a defined serum-free media maybe selected from MCDB 153 medium (Sigma-Aldrich M7403), Modified MCDB153 medium (Biological Industries, Cat. No. 01-059-1), MCDB 105 medium(Sigma-Aldrich M6395), MCDB 110 medium (Sigma-Aldrich M6520), MCDB 131medium (Sigma-Aldrich M8537), MCDB 201 medium (Sigma-Aldrich M6670), andmodified versions thereof. In some embodiments, a defined serum-freemedia is MCDB 153 medium (Sigma-Aldrich M7403). In some embodiments, adefined serum-free media is Modified MCDB 153 medium (BiologicalIndustries, Cat. No. 01-059-1).

In some embodiments, a cell culture medium is a xeno-free serum-freemedium. Xeno-free generally means having no components originating fromanimals other than the animal from which cells being cultured originate.For example, a xeno-free culture has no components of non-human animalorigin when human cells are cultured. In some embodiments, a cellculture medium is a defined xeno-free serum-free medium. Definedxeno-free serum-free media, sometimes referred to as chemically-definedxeno-free serum-free media, generally include identified componentspresent in known concentrations, and generally do not include undefinedcomponents such as animal organ extracts (e.g., pituitary extract) orother undefined animal-derived products (e.g., serum albumin (e.g.,purified from blood), growth factors, hormones, carrier proteins,hydrolysates, and/or attachment factors). Defined xeno-free serum-freemedia may or may not include lipids and/or recombinant proteins fromanimal sources (e.g., non-human sources) such as, for example,recombinant albumin, recombinant growth factors, recombinant insulinand/or recombinant transferrin. Recombinant proteins may be derived, forexample, from non-animal sources such as a plant (e.g., rice) orbacterium (e.g., E. coli), and in certain instances synthetic chemicalsare added to defined media (e.g., a polymer (e.g., polyvinyl alcohol)),which can reproduce some of the functions of bovine serum albumin(BSA)/human serum albumin (HSA). In some embodiments, a definedserum-free medium may comprise a commercially available xeno-free serumsubstitute, such as, for example, XFKOSR™ (Invitrogen). In someembodiments, a defined serum-free medium may comprise a commerciallyavailable xeno-free base medium such as, for example, mTeSR2™ (Stem CellTechnologies), NutriStem™ (StemGent), X-Vivo 10™ or X-Vivo 15™ (LonzaBiosciences), or HEScGRO™ (Millipore).

Additional ingredients may be added to a cell culture medium herein. Forexample, such additional ingredients may include amino acids, vitamins,inorganic salts, inorganic acids, adenine, ethanolamine, D-glucose,heparin, N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid](HEPES), hydrocortisone, insulin, lipoic acid, phenol red,phosphoethanolamine, putrescine, sodium pyruvate, pyruvic acid, ammoniummetavanadate, molybdic acid, silicates, alkali silicates (e.g., sodiummetasilicate), purines, pyrimidines, polyamines, alpha-keto acids,organosulphur compounds, buffers (e.g., HEPES), antioxidants, thiocticacid, triiodothyronine (T3), thymidine and transferrin. In certaininstances, insulin and/or transferrin may be replaced by ferric citrateor ferrous sulfate chelates. Amino acid may include, for example,L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine,L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine,L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,L-threonine, L-tryptophan, L-tyrosine and L-valine. Vitamins mayinclude, for example, biotin, D-biotin, choline chloride,D-Ca⁺²-pantothenate, D-pantothenic acid, folic acid, i-inositol,myo-inositol, niacinamide, pyridoxine, riboflavin, thiamine and vitaminB12. Inorganic salts may include, for example, calcium salt (e.g.,CaCl₂), CuSO₄, FeSO₄, KCl, a magnesium salt (e.g., MgCl₂, MgSO₄), amanganese salt (e.g., MnCl₂), sodium acetate, NaCl, NaHCO₃, Na₂HPO₄,Na₂SO₄, and ions of certain trace elements including selenium, silicon,molybdenum, vanadium, nickel, tin and zinc. These trace elements may beprovided in a variety of forms, including the form of salts such asNa₂SeO₃, Na₂SiO₃, (NH₄)₆Mo₇O₂₄, NH₄VO₃, NiSO₄, SnCl and ZnSO. Additionalingredients may include, for example, heparin, epidermal growth factor(EGF), at least one agent increasing intracellular cyclic adenosinemonophosphate (cAMP) levels, at least one fibroblast growth factor(FGF), acidic FGF, granulocyte macrophage colony-stimulating factor(GM-CSF) (uniprot accession number P04141), granulocyte colonystimulating factor (G-CSF) (uniprot accession number P09919), hepatocytegrowth factor (HGF) (uniprot accession number P14210), neuregulin 1(NRG1) (uniprot accession number Q61CV5), neuregulin 2 (NRG2) (uniprotaccession number Q3MI86), neuregulin 3 (NRG3) (uniprot accession numberB9EGV5), neuregulin 4 (NRG4) (uniprot accession number QOP6N6),epiregulin (ERG) (uniprot accession number 014944), betacellulin (BC)(uniprot accession number Q86UF5), Interleukin-11 (IL11) (uniprotaccession number P20809), a collagen and heparin-binding EGF-like growthfactor (HB-EGF) (uniprot accession number Q14487).

In some embodiments, a cell culture medium comprises calcium. In someembodiments, calcium is present at a concentration of about 2 mM. Insome embodiments, calcium is present at a concentration below 2 mM. Insome embodiments, calcium is present at a concentration of about 1 mM.In some embodiments, calcium is present at a concentration below 1 mM.For example, calcium may be present a concentration below 2 mM, below 1mM, below 900 μM, below 800 μM, below 700 μM, below 600 μM, below 500μM, below 400 μM, below 300 μM, below 200 μM, below 100 μM, below 90 μM,below 80 μM, below 70 μM, below 60 μM, below 50 μM, below 40 μM, below30 μM, below 20 μM, or below 10 μM. In some embodiments, calcium ispresent at a concentration below 500 μM. In some embodiments, calcium ispresent at a concentration below 300 μM. In some embodiments, calcium ispresent at a concentration below 100 μM. In some embodiments, calcium ispresent at a concentration below 20 μM. In some embodiments, calcium ispresent at a concentration of about 90 μM.

In some embodiments, a cell culture medium comprises albumin (e.g.,serum albumin). Albumin is a protein generally abundant in vertebrateblood. In some embodiments, a cell culture medium comprises bovine serumalbumin (BSA). In some embodiments, a cell culture medium compriseshuman serum albumin (HSA). Albumin may be purified (e.g., from human orbovine serum) or may be recombinantly produced, such as for example, inplants (e.g., rice), bacteria (e.g., E. coli), or yeast (e.g., Pichiapastoris, Saccharomyces cerevisiae). In some embodiments, a cell culturemedium comprises recombinant human serum albumin (rHSA). In someembodiments, a cell culture medium comprises recombinant human serumalbumin (rHSA) produced in rice.

In some embodiments, a cell culture medium comprises one or more lipids.Lipids generally refer to oils, fats, waxes, sterols, fat-solublevitamins (e.g., vitamins A, D, E, and K), fatty acids, monoglycerides,diglycerides, triglycerides, phospholipids, glycerolipids,glycerophospholipids, sphingolipids, saccharolipids, polyketides, prenollipids and the like, and may include mixtures of lipids (e.g.,chemically defined lipids mixtures). In some embodiments, lipids may beselected from arachidonic acid, cholesterol, DL-alpha-tocopherolacetate, linoleic acid, linolenic acid, myristic acid, oleic acid,palmitic acid, palmitoleic acid, pluronic F-68, stearic acid,polysorbate 80 (TWEEN 80), TWEEN 20, cod liver oil fatty acids (methylesters), polyoxyethylenesorbitan monooleate, D-α-tocopherol acetate. Insome embodiments, lipids may include one or more of linoleic acid,linolenic acid, oleic acid, palmitic acid, and stearic acid. In someembodiments, a lipids mix may be a commercially available lipids mix(e.g., Chemically Defined Lipid Concentrate (Gibco, 11905-031); LipidMixture (Sigma-Aldrich L5146); Lipid Mixture 1, Chemically Defined(Sigma-Aldrich L0288)). In some embodiments, a lipids mix may include amixture of lipids supplied with a commercially available albumin (e.g.,AlbuMAX® I Lipid-Rich BSA (Gibco, 11020-039)).

In some embodiments, a cell culture medium comprises one or moremitogenic growth factors. For example, a mitogenic growth factor mayinclude epidermal growth factor (EGF), transforming growth factor-alpha(TGF-alpha), fibroblast growth factor (FGF), basic fibroblast growthfactor (bFGF), acidic fibroblast growth factor (aFGF), brain-derivedneurotrophic factor (BDNF), insulin-like growth factor I (IGF-I),insulin-like growth factor II (IGF-II), and/or keratinocyte growthfactor (KGF). In some embodiments, a medium does not comprise amitogenic growth factor.

In some embodiments, a cell culture medium comprises one or moremitogenic supplements. For example, a mitogenic supplement may includebovine pituitary extract (BPE; Gibco/Thermo-Fisher), B27(Gibco/Thermo-Fisher), N-Acetylcysteine (Sigma), GEM21 NEUROPLEX (GeminiBio-Products), and N2 NEUROPLEX (Gemini Bio-Products). In someembodiments, a cell culture medium does not comprise a mitogenicsupplement.

In some embodiments, a cell culture medium comprises one or more agentsthat increase intracellular cyclic adenosine monophosphate (cAMP)levels. For example, a cell culture medium may comprise one or morebeta-adrenergic agonists (e.g., one or more beta-adrenergic receptoragonists). Beta-adrenergic agonists (e.g., beta-adrenergic receptoragonists) generally are a class of sympathomimetic agents which activatebeta adrenoceptors (e.g., beta-1 adrenergic receptor, beta-2 adrenergicreceptor, beta-3 adrenergic receptor). The activation of betaadrenoceptors activates adenylate cyclase, which leads to the activationof cyclic adenosine monophosphate (cAMP). Beta-adrenergic agonists(e.g., beta-adrenergic receptor agonists) may include, for example,epinephrine, isoproterenol, dobutamine, xamoterol, salbutamol(ALBUTEROL), levosalbutamol (LEVALBUTEROL), fenoterol, formoterol,metaproterenol, salmeterol, terbutaline, clenbuterol, isoetarine,pirbuterol, procaterol, ritodrine, arbutamine, befunolol,bromoacetylalprenololmenthane, broxaterol, cimaterol, cirazoline,denopamine, dopexamine, etilefrine, hexoprenaline, higenamine,isoxsuprine, mabuterol, methoxyphenamine, nylidrin, oxyfedrine,prenalterol, ractopamine, reproterol, rimiterol, tretoquinol,tulobuterol, zilpaterol, and zinterol. In some embodiments, a cellculture medium comprises isoproterenol. In some embodiments, a cellculture medium comprises isoproterenol at a concentration of betweenabout 0.5 μM to about 20 μM. For example, isoproterenol may be presentat a concentration of about 0.5 μM, about 0.6 μM, about 0.7 μM, about0.8 μM, about 0.9 μM, about 1 μM, about 1.25 μM, about 1.5 μM, about1.75 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM,about 4.5 μM, about 5 μM, about 5.5 μM, about 6 μM, about 7 μM, about 8μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM,about 14 μM, or about 15 μM.

Other agents that increase intracellular cAMP level may include agentswhich induce a direct increase in intracellular cAMP levels (e.g.,dibutyryl cAMP), agents which cause an increase in intracellular cAMPlevels by an interaction with a cellular G-protein (e.g., cholera toxinand forskolin), and agents which cause an increase in intracellular cAMPlevels by inhibiting the activities of cAMP phosphodiesterases (e.g.,isobutylmethylxanthine (IBMX) and theophylline).

In some embodiments, a cell culture medium does not comprise one or moreof the following: a Wnt agonist, a beta-catenin agonist, Noggin, DAN,Cerberus, Gremlin, R-spondin, Wnt-3a, EGF, nicotinamide, FGF10, gastrin,a p38 inhibitor, SB202190, DHT, a notch inhibitor, a gamma secretaseinhibitor, DBZ, DAPT, Interleukin-6 (IL6), or ephrin A5 (EfnA5).

In some embodiments, a cell culture medium comprises one or moreinhibitors. Inhibitors may include, for example, one or more TGF-betainhibitors (e.g., one or more TGF-beta signaling inhibitors), one ormore p21-activated kinase (PAK) inhibitors, one or more myosin IIinhibitors (e.g., non-muscle myosin II (NM II) inhibitors), and one ormore Rho kinase inhibitors (e.g., one or more Rho-associated proteinkinase inhibitors). Such classes of inhibitors are discussed in furtherdetail below. Inhibitors may be in the form of small molecule inhibitors(e.g., small organic molecules), antibodies, RNAi molecules, antisenseoligonucleotides, recombinant proteins, natural or modified substrates,enzymes, receptors, peptidomimetics, inorganic molecules, peptides,polypeptides, aptamers, and the like and structural or functionalmimetics of these. An inhibitor may act competitively,non-competitively, uncompetitively or by mixed inhibition. For example,in certain embodiments, an inhibitor may be a competitive inhibitor ofthe ATP binding pocket of a target kinase (e.g., protein kinase). Insome embodiments, an inhibitor disrupts the activity of one or morereceptors. In some embodiments, an inhibitor disrupts one or morereceptor-ligand interactions. In some embodiments, an inhibitor may bindto and reduce the activity of its target. In some embodiments, aninhibitor may bind to and reduce the activity of its target by about 10%or more compared to a control. For example, an inhibitor may bind to andreduce the activity of its target by about 20% or more, 30% or more, 40%or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% ormore, 95% or more, or 99% or more compared to a control. Inhibition canbe assessed using a cellular assay, for example.

In some embodiments, an inhibitor is a kinase inhibitor (e.g., a proteinkinase inhibitor). The effectiveness of a kinase inhibitor inhibitingits target's biological or biochemical function may be expressed as anIC₅₀ value. The IC₅₀ generally indicates how much of a particularinhibitor is required to inhibit a kinase by 50%. In some embodiments,an inhibitor has an IC₅₀ value equal to or less than 1000 nM, equal toor less than 500 nM, equal to or less than 400 nM, equal to or less than300 nM, equal to or less than 200 nM, equal to or less than 100 nM,equal to or less than 50 nM, equal to or less than 20 nM, or equal to orless than 10 nM.

In some embodiments, an inhibitor may directly or indirectly affect oneor more cellular activities, functions or characteristics. For example,an inhibitor may induce telomerase reverse transcriptase expression incultured cells, for example through the inhibition of the TGF-betasignaling pathway. In certain embodiments, a TGF-beta inhibitor (e.g., aTGF-beta signaling inhibitor) activates telomerase reverse transcriptaseexpression in cultured cells. In certain embodiments, an ALK5 inhibitoractivates telomerase reverse transcriptase expression in cultured cells.In certain embodiments, A83-01 activates telomerase reversetranscriptase expression in cultured cells. In another example, aninhibitor may modulate the cytoskeletal structure within cultured cells,for example through the inhibition of Rho kinase (e.g., Rho-associatedprotein kinase), p21-activated kinase (PAK), and/or myosin II (e.g.,non-muscle myosin II (NM II)). Modulation the cytoskeletal structure mayinclude, for example, a modification of, a disruption to, or a change inany aspect of cytoskeletal structure including actin microfilaments,tubulin microtubules, and intermediate filaments; or interaction withany associated proteins, such as molecular motors, crosslinkers, cappingproteins and nucleation promoting factors. In certain embodiments, aROCK inhibitor modulates the cytoskeletal structure within culturedcells. In certain embodiments, Y-27632 modulates the cytoskeletalstructure within cultured cells. In certain embodiments, a PAK1inhibitor modulates the cytoskeletal structure within cultured cells. Incertain embodiments, IPA3 modulates the cytoskeletal structure withincultured cells. In certain embodiments, a myosin II inhibitor (e.g., anon-muscle myosin II (NM II) inhibitor) modulates the cytoskeletalstructure within cultured cells. In certain embodiments, blebbistatinmodulates the cytoskeletal structure within cultured cells.

TGF-Beta Inhibitors

In some embodiments, a method herein comprises inhibiting transforminggrowth factor beta (TGF-beta) signaling in cultured epithelial cells.TGF-beta signaling controls proliferation, cellular differentiation, andother functions in a variety of cell types, and can play a role in cellcycle control, regulation of the immune system, and development incertain cell types. Inhibition of TGF-beta signaling may includeinhibition of any TGF-beta signaling pathway and/or member of theTGF-beta superfamily including ligands such as TGF-beta1, TGF-beta2,TGF-beta3, inhibins, activin, anti-müllerian hormone, bone morphogeneticprotein, decapentaplegic and Vg-1; receptors such as TGF-beta type Ireceptor, TGF-beta type II receptor, ALK1, ALK2, ALK3, ALK4, ALK5, ALK6,ALK7 and ALK8; and downstream effectors such as R-SMAD and other SMADproteins (e.g., SMAD1, SMAD2, SMAD3, SMAD4, SMAD5).

In some embodiments, the activity of one or more TGF-beta receptors isinhibited. In some embodiments, one or more TGF-beta receptor-ligandinteractions are inhibited. In some embodiments, a TGF-beta type Ireceptor is inhibited. A TGF-beta type I receptor may include one ormore of ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, ALK7 and ALK8. In someembodiments, the TGF-beta receptor is ALK5.

In some embodiments, a cell culture medium comprises one or moreTGF-beta inhibitors (e.g., one or more TGF-beta signaling inhibitors).In some embodiments, a TGF-beta inhibitor (e.g., a TGF-beta signalinginhibitor) binds to one or more TGF-beta receptors. In some embodiments,a TGF-beta inhibitor (e.g., a TGF-beta signaling inhibitor) binds to oneor more TGF-beta ligands. In some embodiments, a TGF-beta inhibitor(e.g., a TGF-beta signaling inhibitor) binds to one or more SMADproteins. In some embodiments, a TGF-beta inhibitor (e.g., a TGF-betasignaling inhibitor) binds to one or more TGF-beta receptors and one ormore TGF-beta ligands. In some embodiments, a TGF-beta inhibitor (e.g.,a TGF-beta signaling inhibitor) binds to one or more TGF-beta receptorsand one or more SMAD proteins. In some embodiments, a TGF-beta inhibitor(e.g., a TGF-beta signaling inhibitor) disrupts one or more TGF-betareceptor-ligand interactions. In some embodiments, a TGF-beta inhibitor(e.g., a TGF-beta signaling inhibitor) disrupts one or more TGF-betareceptor-SMAD interactions. In some embodiments, a TGF-beta inhibitor(e.g., a TGF-beta signaling inhibitor) blocks phosphorylation orautophosphorylation of a TGF-beta receptor. In some embodiments, aTGF-beta inhibitor (e.g., a TGF-beta signaling inhibitor) promotes thede-phosphorylation of one or more TGF-beta receptors. In someembodiments, a TGF-beta inhibitor (e.g., a TGF-beta signaling inhibitor)blocks phosphorylation of one or more SMAD proteins. In someembodiments, a TGF-beta inhibitor (e.g., a TGF-beta signaling inhibitor)promotes the de-phosphorylation of one or more SMAD proteins. In someembodiments, a TGF-beta inhibitor (e.g., a TGF-beta signaling inhibitor)promotes the ubiquitin-mediated degradation of one or more TGF-betareceptors. In some embodiments, a TGF-beta inhibitor (e.g., a TGF-betasignaling inhibitor) promotes the ubiquitin-mediated degradation of oneor more SMAD proteins. In some embodiments, a TGF-beta inhibitor (e.g.,a TGF-beta signaling inhibitor) affects the nuclear translocation ofSMADs, nuclear shuffling of SMADs, interactions of SMAD withco-activators, and the like. In certain instances, TGF-beta signalingcan be measured by SMAD reporter assays.

A TGF-beta inhibitor (e.g., a TGF-beta signaling inhibitor) may be anALK5 inhibitor, in some embodiments. An ALK5 inhibitor may bind to ALK5or one or more ALK5 ligands or both. An ALK5 inhibitor may bind to ALK5or one or more downstream SMAD proteins or both. An ALK5 inhibitor maydisrupt one or more ALK5-ligand interactions or may disrupt one or moreALK5-SMAD interactions. In some embodiments, an ALK5 inhibitor blocksphosphorylation of SMAD2.

ALK5 inhibitors may include one or more small molecule ALK5 inhibitors.In some embodiments, an ALK5 inhibitor is an ATP analog. In someembodiments, an ALK5 inhibitor comprises the structure of Formula A:

where:

-   -   X, Y and Z independently are chosen from N, C and O;    -   R¹, R² and R³ independently are chosen from hydrogen, C1-C10        alkyl, substituted C1-C10 alkyl, C3-C9 cycloalkyl, substituted        C3-C9 cycloalkyl, C5-C10 aryl, substituted C5-C10 aryl, C5-C10        cycloaryl, substituted C5-C10 cycloaryl, C5-C9 heterocyclic,        substituted C5-C9 heterocyclic, C5-C9 hetercycloaryl,        substituted C5-C9 heterocycloaryl, -linker-(C3-C9 cycloalkyl),        -linker-(substituted C3-C9 cycloalkyl), -linker-(C5-C10 aryl),        -linker-(substituted C5-C10 aryl), -linker-(C5-C10 cycloaryl),        -linker-(substituted C5-C10 cycloaryl), -linker-(C5-C9        heterocyclic), -linker-(substituted C5-C9 heterocyclic),        -linker-(C5-C9 hetercycloaryl), -linker-(substituted C5-C9        heterocycloaryl);    -   n is 0 or 1;    -   R⁴, R⁵ and R⁶ independently are chosen from hydrogen, C1-C10        alkyl, substituted C1-C10 alkyl, C1-C10 alkoxy, substituted        C1-C10 alkoxy, C1-C6 alkanoyl, C1-C6 alkoxycarbonyl, substituted        C1-C6 alkanoyl, substituted C1-C6 alkoxycarbonyl, C3-C9        cycloalkyl, substituted C3-C9 cycloalkyl, C5-C10 aryl,        substituted C5-C10 aryl, C5-C10 cycloaryl, substituted C5-C10        cycloaryl, C5-C9 heterocyclic, substituted C5-C9 heterocyclic,        C5-C9 hetercycloaryl, substituted C5-C9 heterocycloaryl,        -linker-(C3-C9 cycloalkyl), -linker-(substituted C3-C9        cycloalkyl), -linker-(C5-C10 aryl), -linker-(substituted C5-C10        aryl), -linker-(C5-C10 cycloaryl), -linker-(substituted C5-C10        cycloaryl), -linker-(C5-C9 heterocyclic), -linker-(substituted        C5-C9 heterocyclic), -linker-(C5-C9 hetercycloaryl),        -linker-(substituted C5-C9 heterocycloaryl); and    -   the substituents on the substituted alkyl, alkoxy, alkanoyl,        alkoxycarbonyl cycloalkyl, aryl, cycloaryl, heterocyclic or        heterocycloaryl groups are hydroxyl, C1-C10 alkyl, hydroxyl        C1-C10 alkylene, C1-C6 alkoxy, C3-C9 cycloalkyl, C5-C9        heterocyclic, C1-6 alkoxy C1-6 alkenyl, amino, cyano, halogen or        aryl.

ALK5 inhibitors may include, for example, A83-01(3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide),GW788388(4-[4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide),RepSox(2-(3-(6-Methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine), andSB 431542(4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide).In some embodiments, the ALK5 inhibitor is A83-01.

p21-Activated Kinase (PAK) Inhibitors

In some embodiments, a method herein comprises inhibiting the activityof p21-activated kinase (PAK) in cultured epithelial cells. PAKproteins, a family of serine/threonine p21-activated kinases, includePAK1, PAK2, PAK3 and PAK4, and generally function to link the Rho familyof GTPases to cytoskeleton reorganization and nuclear signaling. Theseproteins are targets for Cdc42 and Rac and may function in variousbiological activities. PAK1, for example, can regulate cell motility andmorphology. In some embodiments, a method herein comprises inhibitingthe activity of PAK1 in cultured epithelial cells.

In some embodiments, a cell culture medium comprises one or more PAK1inhibitors. In some embodiments, a PAK1 inhibitor binds to a PAK1protein. In some embodiments, a PAK1 inhibitor binds to one or more PAK1activators (e.g., Cdc42, Rac). In some embodiments, a PAK1 inhibitorbinds to one or more downstream effectors of PAK1. In some embodiments,a PAK1 inhibitor binds to a PAK1 protein and one or more PAK1 activators(e.g., Cdc42, Rac). In some embodiments, a PAK1 inhibitor disrupts oneor more PAK1-activator interactions. In some embodiments, a PAK1inhibitor disrupts one or more PAK1-effector interactions. In someembodiments, a PAK1 inhibitor targets an autoregulatory mechanism andpromotes the inactive conformation of PAK1.

PAK1 inhibitors may include one or more small molecule PAK1 inhibitors.PAK1 inhibitors may include, for example, IPA3(1,1′-Dithiodi-2-naphthtol), AG-1478(N-(3-Chlorophenyl)-6,7-dimethoxy-4-quinazolinanine), FRAX597(6-[2-chloro-4-(1,3-thiazol-5-yl)phenyl]-8-ethyl-2-[4-(4-methylpiperazin-1-yl)anilino]pyrido[2,3-d]pyrimidin-7-one),FRAX486(6-(2,4-Dichlorophenyl)-8-ethyl-2-[[3-fluoro-4-(1-piperazinyl)phenyl]amino]pyrido[2,3-d]pyrimidin-7(8H)-one),and PF-3758309((S)—N-(2-(dimethylamino)-1-phenylethyl)-6,6-dimethyl-3-(2-methylthieno[3,2-d]pyrimidin-4-yl)amino)-4,6-dihydropyrrolo[3,4-c]pyrazole-5(1H)-carboxamide).In some embodiments, the PAK1 inhibitor is IPA3.

Myosin II Inhibitors

In some embodiments, a method herein comprises inhibiting activity ofmyosin II (e.g., non-muscle myosin II (NM II)) in cultured epithelialcells. Myosin II (e.g., non-muscle myosin II (NM II)) is a member of afamily of ATP-dependent motor proteins and plays a role in musclecontraction and other motility processes (e.g., actin-based motility).Non-muscle myosin II (NM II) is an actin-binding protein that has actincross-linking and contractile properties and is regulated by thephosphorylation of its light and heavy chains. Owing to its positiondownstream of convergent signaling pathways, non-muscle myosin II (NMII) is involved in the control of cell adhesion, cell migration andtissue architecture. In higher eukaryotes, non-muscle myosin II isactivated by phosphorylation of its regulatory light chain (MLC) atSer19/Thr18. MLC phosphorylation controls both the assembly of theactomyosin contractile apparatus and its contractility. Two groups ofenzymes generally control MLC phosphorylation. One group includeskinases that phosphorylate MLC (MLC kinases), promoting activity, andthe other is a phosphatase that dephosphorylates MLC, inhibitingactivity. Several kinases can phosphorylate MLC at Ser19/Thr18 in vitroand, in some cases, in vivo. These include, for example, MLCK, ROCK, PAK(p21-activated kinase), citron kinase, ILK (integrin-linked kinase),MRCK (myotonic dystrophyprotein kinase-related, cdc42-binding kinase)and DAPKs (death-associated protein kinases including ZIPK). The majormyosin phosphatase present in smooth and non-muscle cells includes threesubunits: a large subunit of w 130 kDa (referred to as the myosinphosphatase targeting subunit MYPT1 (also called M130/133, M110 orMBS)), a catalytic subunit of 38 kDa (the δ isoform of type 1 proteinphosphatase, PP1c) and a small subunit of 20 kDa. Rho-associate proteinkinase (ROCK) can activate myosin II by inhibiting MYPT1 and by directlyphosphorylating MLC. PAK1 can activate myosin II through thephosphorylation of atypical protein kinase C (aPKCξ).

In some embodiments, a cell culture medium comprises one or more myosinII inhibitors (e.g., non-muscle myosin II (NM II) inhibitors). In someembodiments, a myosin II inhibitor binds to a myosin II protein. In someembodiments, a myosin II inhibitor binds to a myosin head structure. Insome embodiments, a myosin II inhibitor binds to the myosin-ADP-P_(i)complex. In some embodiments, a myosin II inhibitor disrupts myosin IIATPase activity. In some embodiments, a myosin II inhibitor competeswith ATP for binding to myosin II. In some embodiments, a myosin IIinhibitor competes with nucleotide binding to myosin subfragment-1. Insome embodiments, a myosin II inhibitor disrupts myosin II-actinbinding. In some embodiments, a myosin II inhibitor disrupts theinteraction of the myosin head with actin and/or substrate. In someembodiments, a myosin II inhibitor disrupts ATP-induced actomyosindissociation. In some embodiments, a myosin II inhibitor interferes witha phosphate release process. In some embodiments, a myosin II inhibitorprevents rigid actomyosin cross-linking.

Myosin II inhibitors (e.g., non-muscle myosin II (NM II) inhibitors) mayinclude one or more small molecule myosin II inhibitors (e.g., smallmolecule non-muscle myosin II (NM II) inhibitors). Myosin II inhibitorsmay include, for example, blebbistatin((±)-1,2,3,3a-Tetrahydro-3a-hydroxy-6-methyl-1-phenyl-4H-pyrrolo[2,3-b]quinolin-4-one)and analogs thereof (e.g., para-nitroblebbistatin,(S)-nitro-Blebbistatin, S-(−)-7-desmethyl-8-nitro blebbistatin, and thelike), BTS (N-benzyl-p-toluene sulphonamide), and BDM (2,3-butanedionemonoxime). In some embodiments, the myosin II inhibitor is blebbistatin.

ROCK (Rho-Associated Protein Kinase) Inhibitors

In some embodiments, a method herein comprises inhibiting the activityof Rho kinase (e.g., Rho-associated protein kinase) in culturedepithelial cells. In some embodiments, a method herein does not compriseinhibiting the activity of Rho kinase (e.g., Rho-associated proteinkinase) in cultured epithelial cells. Rho kinase (e.g., Rho-associatedprotein kinase) belongs to the Rho GTPase family of proteins, whichincludes Rho, Rac1 and Cdc42 kinases. An effector molecule of Rho isROCK, which is a serine/threonine kinase that binds to the GTP-boundform of Rho. The catalytic kinase domain of ROCK, which comprisesconserved motifs characteristic of serine/threonine kinases, is found atthe N-terminus. ROCK proteins also have a central coiled-coil domain,which includes a Rho-binding domain (RBD). The C-terminus contains apleckstrin-homology (PH) domain with an internal cysteine-rich domain.The coiled-coil domain is thought to interact with other alpha helicalproteins. The RBD, located within the coiled-coil domain, interacts withactivated Rho GTPases, including RhoA, RhoB, and RhoC. The PH domain isthought to interact with lipid mediators such as arachidonic acid andsphingosylphosphorylcholine, and may play a role in proteinlocalization. Interaction of the PH domain and RBD with the kinasedomain results in an auto-inhibitory loop. In addition, the kinasedomain is involved in binding to RhoE, which is a negative regulator ofROCK activity.

The ROCK family includes ROCK1 (also known as ROK-beta or p160ROCK) andROCK2 (also known as ROK-alpha). ROCK1 is about 1354 amino acids inlength and ROCK2 is about 1388 amino acids in length. The amino acidsequences of human ROCK1 and human ROCK2 can be found at UniProtKnowledgebase (UniProtKB) Accession Number Q13464 and 075116,respectively. The nucleotide sequences of human ROCK1 and ROCK2 can befound at GenBank Accession Number NM_005406.2 and NM_004850,respectively. The nucleotide and amino acid sequences of ROCK1 and ROCK2proteins from a variety of animals can be found in both the UniProt andGenBank databases.

Although both ROCK isoforms are ubiquitously expressed in tissues, theyexhibit differing intensities in some tissues. For example, ROCK2 ismore prevalent in brain and skeletal muscle, while ROCK1 is moreabundant in liver, testes and kidney. Both isoforms are expressed invascular smooth muscle and heart. In the resting state, both ROCK1 andROCK2 are primarily cytosolic, but are translocated to the membrane uponRho activation. Rho-dependent ROCK activation is highly cell-typedependent, and ROCK activity is regulated by several differentmechanisms including changes in contractility, cell permeability,migration and proliferation to apoptosis. Several ROCK substrates havebeen identified (see e.g., Hu and Lee, Expert Opin. Ther. Targets9:715-736 (2005); Loirand et al, Cir. Res. 98:322-334 (2006); and Rientoand Ridley, Nat. Rev. Mol. Cell Bioi. 4:446-456 (2003) all of which areincorporated by reference). In some instances, ROCK phosphorylates LIMkinase and myosin light chain (MLC) phosphatase after being activatedthrough binding of GTP-bound Rho.

Inhibiting the activity of Rho kinase (e.g., Rho-associated proteinkinase) may include reducing the activity, reducing the function, orreducing the expression of at least one of ROCK1 or ROCK2.

The activity, function or expression may be completely suppressed (i.e.,no activity, function or expression); or the activity, function orexpression may be lower in treated versus untreated cells. In someembodiments, inhibiting the activity of Rho kinase (e.g., Rho-associatedprotein kinase) involves blocking an upstream effector of a ROCK1 and/orROCK2 pathway, for example GTP-bound Rho, such that ROCK1 and/or ROCK2are not activated or its activity is reduced compared to untreatedcells. Other upstream effectors include but are not limited to,integrins, growth factor receptors, including but not limited to,TGF-beta and EGFR, cadherins, G protein coupled receptors and the like.In some embodiments, inhibiting the activity of Rho kinase (e.g.,Rho-associated protein kinase) involves blocking the activity, functionor expression of downstream effector molecules of activated ROCK1 and/orROCK2 such that ROCK1 and/or ROCK2 cannot propagate any signal or canonly propagate a reduced signal compared to untreated cells. Downstreameffectors include but are not limited to, vimentin, LIMK, Myosin lightchain kinase, NHEI, cofilin and the like.

In some embodiments, inhibiting the activity of Rho kinase (e.g.,Rho-associated protein kinase) may comprise the use of one or more Rhokinase inhibitors (e.g., one or more Rho-associated protein kinaseinhibitors). Rho kinase inhibitors (e.g., Rho-associated protein kinaseinhibitors) may include one or more small molecule Rho kinase inhibitors(e.g., one or more small molecule Rho-associated protein kinaseinhibitors). Examples of molecule Rho kinase inhibitors (e.g.,Rho-associated protein kinase inhibitors) include, for example, Y-27632((R)-(+)-trans-4-(1-Aminoethyl)-N-(4-Pyridyl)cyclohexanecarboxamidedihydrochloride), SR 3677(N-[2-[2-(Dimethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl-2,3-dihydro-1,4-benzodioxin-2-carboxamidedihydrochloride), thiazovivin(N-Benzyl-[2-(pyrimidin-4-yl)amino]thiazole-4-carboxamide), HA1100hydrochloride(1-[(1,2-Dihydro-1-oxo-5-isoquinolinyl)sulfonyl]hexahydro-1H-1,4-diazepinehydrochloride), HA1077 (fasudil hydrochloride), and GSK-429286(4-[4-(Trifluoromethyl)phenyl]-N-(6-Fluoro-1H-indazol-5-yl)-2-methyl-6-oxo-1,4,5,6-tetrahydro-3-pyridinecarboxamide),each of which is commercially available. Additional small molecule Rhokinase inhibitors (e.g., small molecule Rho-associated protein kinaseinhibitors) include those described, for example, in InternationalPatent Application Publication Nos. WO 03/059913, WO 03/064397, WO05/003101, WO 04/112719, WO 03/062225 and WO 03/062227, and described inU.S. Pat. Nos. 7,217,722 and 7,199,147, and U.S. Patent ApplicationPublication Nos. 2003/0220357, 2006/0241127, 2005/0182040 and2005/0197328, the contents of all of which are incorporated byreference.

Subsequent Environments

In some embodiments, the cells may be removed from the cultureconditions described above after a certain amount of time and placedinto a subsequent environment. Any of the components described above maybe absent in a subsequent environment. In some embodiments, one or moreinhibitors described above is absent in a subsequent environment. Forexample, one or more of a TGF-beta inhibitor (e.g., a TGF-beta signalinginhibitor), ROCK inhibitor, PAK1 inhibitor and a myosin II inhibitor(e.g., non-muscle myosin II (NM II) inhibitor) may be absent in asubsequent environment.

A subsequent environment may be an environment that promotesdifferentiation of the cells. A subsequent environment may be an in vivoenvironment that is similar or identical to the organ or tissue fromwhich the cells were originally derived (e.g., an autologous implant). Asubsequent environment may be an in vitro or ex vivo environment thatclosely resembles certain biochemical or physiological properties of theorgan or tissue from which the cells were originally derived. Asubsequent environment may be a synthetic environment such that factorsknown to promote differentiation in vitro or ex vivo are added to thecell culture. For example, calcium or additional calcium may be added tothe cell culture to promote differentiation. In some embodiments,calcium may be added such that the calcium concentration in the cellculture medium is at least about 1 mM to promote differentiation. Forexample, the calcium concentration in the cell culture medium can be atleast about 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7mM, 1.8 mM, 1.9 mM or 2.0 mM. In some embodiments, calcium is added to acell culture such that the calcium concentration in the cell culturemedium is about 1.5 mM to promote differentiation.

In some embodiments, cells are placed into a subsequent environment thatis specific to stimulate differentiation of cells into the cells of theorgan or tissue from which the cells were originally derived. In someembodiments, cells can be seeded onto one side of a permeable membrane.In some embodiments, cells cultured on one side of a permeable membranecan be exposed to air while the cells receive nutrients from the otherside of the permeable membrane, and such culture may be referred to asan air-liquid-interface. In some instances, cells develop increasingtransmembrane electric resistance (TEER) during air-liquid-interfacedifferentiation. In some embodiments, cells can be seeded in asubsequent environment into or onto a natural or syntheticthree-dimensional cell culture surface. A non-limiting example of athree-dimensional surface is a Matrigel®-coated culture surface. In someembodiments, the cells can be embedded in Matrigel® or other hydrogels.Other three dimensional culture environments include surfaces comprisingcollagen gel and/or a synthetic biopolymeric material in anyconfiguration, such as a hydrogel, for example.

In some embodiments, epithelial cells form tight junctions in culture.Tight junctions generally are parts of cell membranes joined together toform an impermeable or substantially impermeable barrier to fluid.Formation of tight junctions may be visualized, for example, byimmunofluorescence staining of tight junction proteins (e.g., ZO-1). Insome embodiments, epithelial cells can be induced to form tightjunctions in culture. For example, epithelial cells can be induced toform tight junctions when exposed to certain concentrations of calcium.In some embodiments, epithelial cells can be induced to form tightjunctions when exposed to calcium concentrations that are about 1 mM orhigher. For example, epithelial cells can be induced to form tightjunctions when exposed to calcium concentrations that are about 1 mM,1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM,2.0 mM, or higher. In some embodiments, epithelial cells can be inducedto form tight junctions when exposed to a calcium concentration of about1.5 mM.

In some embodiments, epithelial cells form domes or dome-like structuresin culture. Domes generally are multicellular hemicyst structures uniqueto polarized epithelia in culture and can be functionally equivalent todifferentiated epithelium with trans-epithelial solute transport. Domescan occur sporadically in small areas during cell confluence, and oftenmark the initial differentiation process of a functional epithelialmonolayer. In certain instances, dome formation may include one or moreof expression of tight junction proteins, impermeable substratumformation, and diminished cellular adherence to an underlying support(e.g., as a result of liquid accumulation between the cell layer and theunderlying support). Dome formation may occur, for example, duringdevelopment of transepithelial transport systems for morphologicallypolarized cells (see e.g., Su et al. (2007) J. Biol. Chem.282(13):9883-9894). In some embodiments, epithelial cells can be inducedto form domes or dome-like structures in culture. For example,epithelial cells can be induced to form domes or dome-like structureswhen exposed to certain concentrations of calcium. In some embodiments,epithelial cells can be induced to form domes or dome-like structureswhen exposed to calcium concentrations that are about 1 mM or higher.For example, epithelial cells can be induced to form domes or dome-likestructures when exposed to calcium concentrations that are about 1 mM,1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM,2.0 mM, or higher. In some embodiments, epithelial cells can be inducedto form domes or dome-like structures when exposed to a calciumconcentration of about 1.5 mM.

In some embodiments, the cells are placed into a subsequent environmentwhere TGF-beta signaling is not inhibited. In some embodiments, thecells are placed into a subsequent environment where ROCK is notinhibited. In some embodiments, the cells are placed into a subsequentenvironment where PAK1 is not inhibited. In some embodiments, the cellsare placed into a subsequent environment where myosin II (e.g.,non-muscle myosin II (NM II)) is not inhibited. In some embodiments, thecells are placed into a subsequent environment where TGF-beta signalingand ROCK are not inhibited. In some embodiments, the cells are placedinto a subsequent environment where TGF-beta signaling and PAK1 are notinhibited. In some embodiments, the cells are placed into a subsequentenvironment where TGF-beta signaling and myosin II (e.g., non-musclemyosin II (NM II)) are not inhibited.

In some embodiments, the cells maintain or regain one or more nativefunctional characteristics after placement into the cell cultureenvironment where TGF-beta signaling is not inhibited. In someembodiments, the cells maintain or regain one or more native functionalcharacteristics after placement into the cell culture environment whereROCK is not inhibited. In some embodiments, the cells maintain or regainone or more native functional characteristics after placement into thecell culture environment where PAK1 is not inhibited. In someembodiments, the cells maintain or regain one or more native functionalcharacteristics after placement into the cell culture environment wheremyosin II (e.g., non-muscle myosin II (NM II)) is not inhibited. In someembodiments, the cells maintain or regain one or more native functionalcharacteristics after placement into the cell culture environment whereTGF-beta signaling and ROCK are not inhibited. In some embodiments, thecells maintain or regain one or more native functional characteristicsafter placement into the cell culture environment where TGF-betasignaling and PAK1 are not inhibited. In some embodiments, the cellsmaintain or regain one or more native functional characteristics afterplacement into the cell culture environment where TGF-beta signaling andmyosin II (e.g., non-muscle myosin II (NM II)) are not inhibited.

Uses of Expanded Cells

In certain embodiments, an expanded epithelial cell population may beused for certain biomedical and laboratory uses such as, for example,biomolecule production (e.g., protein expression), diagnostics (e.g.,identifying abnormal epithelial cells) and/or therapeutics (e.g.,screening candidate therapeutic agents; cell therapy (e.g., geneticallymodified cells for cell therapy)). In some instances, an expandedepithelial cell population may be used for autologous applications(e.g., autologous implant), and in certain instances, an expandedepithelial cell population may be used for non-autologous applications(e.g., drug screening). In some instances, an expanded epithelial cellpopulation may be collected and/or isolated and/or stored (e.g., for acell bank).

In one example, an expanded epithelial cell population may be used forprotein expression, virus/vaccine production, and the like. In someinstances, an expanded epithelial cell population can be geneticallymodified to express a protein of interest (e.g., a therapeutic protein).In some instances, an epithelial cell or group of cells can begenetically modified and then expanded using expansion conditionsdescribed herein. Such genetic modification of the cells generally wouldnot be a modification intended to increase cell expansion. Rather, suchgenetic modification of the cells would be designed to, for example,insert a transgene (e.g., a disease-modifying transgene) that codes fora particular protein. A protein expressed by a transgene may act as afunctional version of a missing or a defective protein, or may act as asuppressor or inhibitor of genes or other proteins. Cells expressing aparticular protein can then be placed in a subsequent environment, forexample, such as an autologous implant into a subject, such that thecells will produce the protein in vivo

In another example, an expanded epithelial cell population can be usefulfor identifying candidate treatments for a subject having a conditionmarked by the presence of abnormal or diseased epithelial cells. Suchconditions may include for example neoplasias, hyperplasias, andmalignant tumors or benign tumors. In some instances, abnormalepithelial cells obtained from a subject may be expanded according toany of the expansion conditions described herein to produce an in vitropopulation of abnormal epithelial cells. For example, circulating tumorcells (CTCs) may be isolated from a subject's circulation, and theexpansion conditions herein may be utilized to obtain a sufficientnumber of cells for further analysis, such as, for example, functional,phenotypic and/or genetic characterization of the cells.

In another example, an expanded epithelial cell population may be usefulfor identifying one or more candidate treatments for a subject. Forexample, an expanded epithelial cell population may be assayed forgenerating a response profile. A response profile typically is acollection of one or more data points that can indicate the likelihoodthat a particular treatment will produce a desired response, for examplein normal or abnormal epithelial cells. A response to a therapeuticagent may include, for example, cell death (e.g., by necrosis, toxicity,apoptosis, and the like), and/or a reduction of growth rate for thecells. Methods to assess a response to a therapeutic agent include, forexample, determining a dose response curve, a cell survival curve, atherapeutic index and the like. For example, nasal or trachea epithelialcells may be isolated from a subject carrying mutation(s) in the CFTRgene, and the expansion conditions herein may be utilized to obtain asufficient number of cells for further analysis, such as, for example,assays for generating a response profile to therapeutic agents such asdrugs and/or antibodies.

In another example, an expanded epithelial cell population may be usefulfor identifying one or more abnormal epithelial cells in a subject. Forexample, at least one candidate abnormal epithelial cell may be expandedaccording to any of the expansion conditions described herein. Once thecells have been expanded, for example, a tissue origin profile can bedetermined (e.g., by assaying mRNA and/or protein expression,histological evaluation, immunohistochemical staining) for the cells todetermine the likely tissue of origin. At least one feature of the cellscan be compared to the same feature of normal epithelial cells from thesame tissue of origin. Cell features that may be compared include, forexample, cell growth characteristics, colony formation, proteomicprofiles, metabolic profiles and genomic profiles. A detected differencein the candidate abnormal epithelial cells and the normal epithelialcells may indicate that the candidate abnormal epithelial cells areabnormal compared to normal epithelial cells.

In another example, an expanded epithelial cell population may be usefulfor monitoring the progression of a disease or treatment of a disease ina subject. Monitoring the progression of a disease generally meansperiodically checking an abnormal condition in a subject to determine ifan abnormal condition is progressing (worsening), regressing(improving), or remaining static (no detectable change). Expandedepithelial cells from a subject may be assayed for various markers ofprogression or regression. Monitoring the progression of a disease alsomay include monitoring the efficacy of one or more treatments.

EXAMPLES

The examples set forth below illustrate certain embodiments and do notlimit the technology.

Example 1 Materials and Methods

The materials and methods set forth in this Example were used to performcell culture and other assays described in Examples 2 to 9, except whereotherwise noted.

Cell Culture and Determination of Population Doublings

Epithelial cells (prostate epithelial cells and bronchial epithelialcells) were plated at 3,000-10,000 viable cells/cm² in tissue culturevessels, using culture medium as indicated in Examples 2 to 5 and FIGS.1 to 17, and incubated at 37° C. with 5% CO₂. The medium was changedevery 2 or 3 days. Cells were sub-cultured using standard trypsinizationmethod when they were about 70-90% confluent. Total cell number wasdetermined using the Countess II Automated Cell Counter (LifeTechnologies, AMQAX1000) following manufacturer's instructions.

Cells and media used for these assays included: PrEC Prostate EpithelialCells (Lonza CC-2555); Normal human bronchial epithelial cells (LonzaCC-2540); LNCap Clone FGC Cell Line (Sigma-Aldrich, D-073); PrEGMBulletKit containing PrEBM Basal Medium and PrEGM SingleQuot KitSupplements & Growth Factors (Lonza CC-3166); and Keratinocyte-SFM(Gibco/Thermo-Fisher 17005-042) supplied with prequalified humanrecombinant Epidermal Growth Factor 1-53 (EGF 1-53, used at 0.2 ng/mL)and Bovine Pituitary Extract (BPE, used at 30 μg/mL). Cell culturematerials included Corning® BioCoat™ Cellware, Collagen Type I, T-25flask (Corning, 356484).

A formula used for the calculation of population doublings (PD) ispresented in Equation A:

n=3.32*(log Y−log I)+X  Equation A

where n=the final PD number at end of a given subculture, Y=the cellyield at the time of harvesting, I=the cell number used as inoculum tobegin that subculture, and X=the doubling level of the inoculum used toinitiate the subculture being quantitated.

Stocks of certain chemicals used in the study were prepared bydissolving in DMSO to 10 mM. The chemical stocks were added to culturemedia to desired final concentrations, as described in Examples 2 to 5and shown in FIGS. 1 to 17, from the time when cell culture wasinitiated. Certain compounds used are listed in Table 1 below.

TABLE 1 Listing of compounds Concentrations Chemical Name TargetSupplier Cat # used A 83-01 TGF-beta RI, ALK4 and Sigma-Aldrich, 0.1-10μM  ALK7 SML0788; Tocris 2939 SB 431542 TGF-beta RI, ALK4 and Tocris1614 0.1-5 μM ALK7 RepSox TGF-beta RI, ALK4 and Tocris 3742 0.1-5 μMALK7 GW 788388 TGF-beta RI, ALK4 and Tocris 3264 0.1-5 μM ALK7 Y-27632Rho-kinase (Rho- Enzo Life Sciences, 0.1-10 μM  associated proteinkinase, ALX-270-333-M025 ROCK) SR 3677 Rho-kinase (Rho- Tocris 36670.1-5 μM dihydrochloride associated protein kinase, ROCK) GSK 429286Rho-kinase (Rho- Tocris 3726 0.1-5 μM associated protein kinase, ROCK)Thiazovivin Rho-kinase (Rho- Tocris 3845 0.1-5 μM associated proteinkinase, ROCK) IPA-3 Group I p21-activated Tocris 3622 0.1-5 μM kinase(PAK) Blebbistatin myosin II ATPase (i.e., Tocris 1760 0.1-5 μMnon-muscle myosin II (NM II) ATPase) Isoproterenol β-adrenoceptoragonist Sigma-Aldrich I5627 0.1-5 μM

Quantitative RT-PCR

Total RNA was prepared using TRIzol® Plus RNA Purification Kit (LifeTechnologies, 12183-555) and PureLink® RNA Mini Kit (Life Technologies,12183018A), following the manufacturer's instructions. One hundrednanogram total RNA was used for the determination of human telomerasereverse transcriptase (hTERT) expression using the TaqMan® RNA-to-CT™1-Step Kit (Life Technologies, 4392938), following the protocol providedby the supplier. hTERT primers and Taqman probe used were as follows:forward primer, 5′-TGACACCTCACCTCACCCAC-3′ (SEQ ID NO:1), reverseprimer, 5′-CACTGTCTTCCGCAAGTTCAC-3′ (SEQ ID NO:2) and Taqman probe,5′-ACCCTGGTCCGAGGTGTCCCTGAG-3′ (SEQ ID NO:3).

Example 2 Growth of Epithelial Cells in Conventional Cell Culture Medium(i.e., Control Culture Conditions)

In this example, prostate epithelial cells (PrEC) and bronchialepithelial cells (HBEC) were grown in conventional cell culture medium(i.e., control culture conditions) to demonstrate certain properties ofepithelial cell growth in vitro and/or ex vivo.

Prostate epithelial cells (PrEC) were cultured in one of two types ofregular culture media generally used for culturing prostate epithelialcells: 1) Prostate Epithelial Cell Growth Medium (PrEGM BulletKitcontaining PrEBM Basal Medium and PrEGM SingleQuot Kit Supplements &Growth Factors (Lonza CC-3166)), or 2) KSFM (Keratinocyte-SFM(Gibco/Thermo-Fisher 17005-042) supplied with prequalified humanrecombinant Epidermal Growth Factor 1-53 (EGF 1-53, used at 0.2 ng/mL)and Bovine Pituitary Extract (BPE, used at 30 μg/mL)). Populationdoublings of the cells in each passage were calculated, and totalpopulation doublings were plotted against number of days of culture(FIG. 1, top panel). In both media types, prostate epithelial cellsshowed limited cell replication of only 10 to 20 population doublingsbefore entering senescence. Prostate epithelial cells cultured in KSFMexhibited morphology characteristic of cell senescence at populationdoubling 18 (PD 18; FIG. 1, bottom panel).

Bronchial epithelial cells (HBEC) were cultured in KSFM. Populationdoublings of the cells in each passage were calculated, and totalpopulation doublings were plotted against number of days of culture(FIG. 2, top panel). The bronchial epithelial cells showed activereplication for only 11 population doublings before entering cellsenescence. Bronchial epithelial cells cultured in KSFM exhibitedcharacteristic morphology of cell senescence at population doubling 11(PD 11; FIG. 2, bottom panel).

Expression of human telomerase reverse transcriptase (hTERT) gene wasexamined by quantitative real-time PCR in bronchial epithelial cells andprostate epithelial cells at different passages. The ends of chromosomesare composed of repeated segments of DNA structures called telomeres,which protect chromosomes from abnormally sticking together or breakingdown (degrading). In normal epithelial cells, telomeres typically becomeprogressively shorter as the cell divides due to the lack of expressionof telomerase reverse transcriptase (TERT). Telomerase is generallyactive in stem cells and abnormally active in most cancer cells, whichgrow and divide without limitation. The level of hTERT expression wascompared to LNCaP cells, a human prostate cancer cell line(Sigma-Aldrich, D-073), which was used as positive control for hTERTexpression. The bronchial epithelial cells did not express hTERT ateither early (p3) or late (p5) passages. The prostate epithelial cellsat early passage (p2) expressed extremely low level of hTERT, whichquickly diminished at passage 3 and become non-detected by passage 5(FIG. 3A and FIG. 3B). These findings confirmed that both PrEC and HBECare normal epithelial cells.

Expression of certain cell markers was examined in cultured prostateepithelial cells. These cells did not express an epithelial stem cellmarker, Lgr5, as examined by immunofluorescence staining (FIG. 4, bottompanel). Instead, all cells stained positive for TP63 expression (FIG. 4,middle panel). The TP63 gene, a transcription factor, is a marker ofbasal epithelial cells and generally is required for normal function ofepithelial tissues. Cell nuclei were visualized using DAPI staining(FIG. 4, top panel). Polyclonal Lgr5 antibody (Anti-GPR49/LGR5 Antibody,LifeSpan Biosciences, LS-A1235) was used at 1:50 dilution. Monoclonalanti-TP63 antibody (Santa Cruz Biotechnology, sc-25268) was used at 1:50dilution.

The experiments above therefore demonstrated that epithelial cellscultured in conventional cell culture media (i.e., control cultureconditions) had limited proliferation capacity in vitro and/or ex vivo,extremely low or undetected expression of the telomerase reversetranscriptase gene, and did not express protein markers typical ofepithelial stem cells (i.e., did not express the epithelial stem cellmarker LGR5).

Example 3 Human Telomerase Reverse Transcriptase (hTERT) Expression inthe Presence of an ALK5 Inhibitor

In this example, hTERT expression in epithelial cells was assessed inthe presence of an ALK5 inhibitor, a Rho kinase inhibitor (i.e., aRho-associated protein kinase inhibitor), or both. Expression of hTERTgene was examined by quantitative real-time PCR in bronchial epithelialcells and prostate epithelial cells. The cells were treated with an ALK5inhibitor, A83-01, or a Rho kinase inhibitor (i.e., a Rho-associatedprotein kinase inhibitor), Y-27632, or both inhibitors, at differentpassages in KSFM. A83-01 quickly induced and sustained hTERT expressionin epithelial cells as described below.

As shown in FIG. 5A and FIG. 5B, bronchial epithelial cells did notexpress hTERT at both early (p3) and late (p5) passages in KSFM. ALK5inhibitor A83-01 robustly induced hTERT expression in bronchialepithelial cells at early passage (p3), and sustained measurable hTERTexpression at late passage (p6). Rho kinase inhibitor (i.e.,Rho-associated protein kinase inhibitor) Y-27632 slightly induced hTERTexpression in bronchial epithelial cells at early passage (p3); however,it became non-detectable at late passage (p6). In the presence of bothA83-01 and Y-27632, hTERT expression was induced and sustained at latepassage (p6) in bronchial epithelial cells.

As shown in FIG. 6A and FIG. 6B, prostate epithelial cells at earlypassage (p2) expressed low level of hTERT, which quickly diminished atpassage 3 and became non-detectable by passage 5. ALK5 inhibitor A83-01quickly induced hTERT expression in prostate epithelial cells at earlypassage (p2), and sustained measurable hTERT expression at late passage(p5). Rho kinase inhibitor (i.e., Rho-associated protein kinaseinhibitor) Y-27632 induced hTERT expression in prostate epithelial cellsat early passage (p2); however, it became non-detectable at late passage(p5). In the presence of both A83-10 and Y-27632, hTERT expression wasinduced significantly in early passage (p2) prostate epithelial cellsand sustained at high level even at late passage (p5). This level ofhTERT expression was comparable to the level of hTERT expression inprostate epithelial cells co-cultured with 3T3-J2 feeder cells (J2).

Example 4 Epithelial Cell Plating Efficiency

In this example, epithelial cell plating efficiency, i.e., number ofcells that efficiently attach to the cell culture surfaces, continue todivide and grow into colonies, was examined under various conditions.

As shown in FIG. 7, ALK5 inhibitor A83-01 interfered with prostateepithelial cell plating efficiency on regular tissue culture surface(i.e., uncoated) in KSFM. In the presence of ALK5 inhibitor A83-01, mostprostate epithelial cells failed to attach to the regular tissue culturesurface even after 3 days. However, those few cells that did attachcontinued to proliferate. By day 8, when the cells were passaged, mostcells exhibited characteristic morphology of actively dividing cells.

As shown in FIG. 8, ALK5 inhibitor A83-01 interfered with bronchialepithelial cell plating efficiency on regular tissue culture surface(i.e., uncoated) in KSFM. In the presence of ALK5 inhibitor A83-01, mostbronchial epithelial cells failed to attach to the regular tissueculture surface even after 3 days. However, those few cells that didattach continued to proliferate. Most cells exhibited characteristicmorphology of actively dividing cells at day 7.

As shown in FIG. 9, Rho kinase inhibitor (i.e., Rho-associated proteinkinase inhibitor) Y-27632 ameliorated the low plating efficiency ofprostate epithelial cells caused by A83-01 in KSFM. Many prostateepithelial cells attached to regular tissue culture surface in thepresence of both A83-01 and Y-27632 at day 2 after plating. The cellscontinued to proliferate and most cells exhibited characteristicmorphology of actively dividing cells at day 7.

As shown in FIG. 10, Rho kinase inhibitor (i.e., Rho-associated proteinkinase inhibitor) Y-27632 ameliorated the low plating efficiency ofbronchial epithelial cells caused by A83-01 in KSFM. Many bronchialepithelial cells attached to regular tissue culture surface in thepresence of both A83-01 and Y-27632 at day 2 after plating. The cellscontinued to proliferate and most cells exhibited characteristicmorphology of actively dividing cells at day 7.

As shown in FIG. 11, use of collagen I-coated tissue culture surfacedramatically increased the plating efficiency of prostate epithelialcells in the presence of A83-01 and Y-27632 in KSFM. Most cellsefficiently attached to the surface at day 1 after plating, andproliferated quickly. By day 5, when the cells were passaged, most cellsexhibited characteristic morphology of actively dividing cells.

As shown in FIG. 12, use of collagen I-coated tissue culture surfacedramatically increased the plating efficiency of bronchial epithelialcells in the presence of A83-01 and Y-27632 in KSFM. Most cellsefficiently attached to the surface at day 1 after plating, andproliferated quickly. By day 6, when the cells were passaged, most cellsexhibited characteristic morphology of actively dividing cells.

Example 5 The Effects of Compounds Including ALK5 Inhibitors, Rho KinaseInhibitors, and Other Compounds on Epithelial Cell Proliferation

In this example, proliferation of prostate and bronchial epithelialcells was assessed in the presence of compounds including ALK5inhibitors, Rho kinase inhibitors (i.e., Rho-associated protein kinaseinhibitors), and other compounds.

Prostate and bronchial epithelial cells were grown in KSFM in thepresence of Rho kinase inhibitor (i.e., Rho-associated protein kinaseinhibitor) Y-27632. As shown in FIG. 13, both prostate and bronchialepithelial cells entered senescence at late passage despite thecontinuous use of Rho kinase inhibitor (i.e., Rho-associated proteinkinase inhibitor) Y-27632. By day 8 or day 9, late passages of prostateepithelial cells and bronchial epithelial cells exhibited characteristicmorphology of cell senescence such as a flat and enlarged cell shape.Thus, when it was used alone, Y-27632 did not promote the proliferationof prostate epithelial cells or bronchial epithelial cells.

To examine the effects of additional in vitro and/or ex vivo growthconditions, prostate epithelial cells were grown in KSFM or PrEGM in thepresence of media alone, ALK5 inhibitor A83-01, Rho kinase inhibitor(i.e., Rho-associated protein kinase inhibitor) Y-27632, or both (withor without collagen). As shown in FIG. 14, prostate epithelial cellsentered senescence after 10 to 20 population doublings in PrEGM or KSFM.Rho kinase inhibitor (i.e., Rho-associated protein kinase inhibitor)Y-27632 slightly increased the total population doublings of prostateepithelial cells, although the cells still entered senescence shortlyafter reaching over PD20. A83-01 also increased the total populationdoublings; however, since A83-01 can interfere with the platingefficiency on regular tissue culture surface (see Example 4), theincrease in cell number is slow. Adding both A83-01 and Y-27632 to KSFMsignificantly increased the total population doublings of prostateepithelial cells at a much faster pace, on both regular tissue culturevessel and collagen-coated tissue culture vessels. Thus, totalpopulation doublings of prostate epithelial cells increasedsignificantly when ALK5 inhibitor A83-01 and Rho kinase inhibitor (i.e.,Rho-associated protein kinase inhibitor) Y-27632 were used incombination.

Similar to the experiment above, bronchial epithelial cells were grownin KSFM in the presence of media alone, ALK5 inhibitor A83-01, Rhokinase inhibitor (i.e., Rho-associated protein kinase inhibitor)Y-27632, or both (with or without collagen). As shown in FIG. 15,bronchial epithelial cells entered senescence after 11 populationdoublings in KSFM. Rho kinase inhibitor (i.e., Rho-associated proteinkinase inhibitor) Y-27632 slightly increased the total populationdoublings of bronchial epithelial cells, although the cells stillentered senescence shortly after reaching over PD13. A83-01 alsoincreased the total population doublings; however, since A83-01 caninterfere with the plating efficiency on regular tissue culture surface(see Example 4), the increase in cell number is slow. Adding both A83-01and Y-27632 to KSFM significantly increased the total populationdoublings of bronchial epithelial cells at a much faster pace, on bothregular tissue culture surfaces and collagen-coated tissue culturesurfaces, the latter of which showed an even faster pace. Thus, totalpopulation doublings of bronchial epithelial cells increasedsignificantly when ALK5 inhibitor A83-01 and Rho kinase inhibitor (i.e.,Rho-associated protein kinase inhibitor) Y-27632 were used incombination.

In another experiment, late passage prostate epithelial cells werecultured in KSFM plus individual compounds as indicated in FIG. 16,which were tested at various concentrations (i.e., 5 μM, filled bar; 1μM, checkered bar; and 0.2 μM, open bar). In a control experiment withno added compound, there was minimum increase of cell numbers after 5days. Rho kinase inhibitors (i.e., Rho-associated protein kinaseinhibitors) such as Y-27632, SR 3677, GSK 429286 and Thiazovivin lead toslight increase of total cell numbers. In contrast, ALK5 inhibitors suchas A83-01, SB 431542, GW 788388 and RepSox resulted in a pronouncedincrease of total cell number after 5 days. Neither PAK1 inhibitor IPA-3nor myosin II inhibitor (i.e., non-muscle myosin II (NM II) inhibitor)Blebbistatin resulted in any increase of cell proliferation, and IPA-3caused nearly total cell death at the highest concentration tested (5μM). Thus, when used alone, ALK5 inhibitors significantly increased theproliferation of late-passage prostate epithelial cells in KSFM; whenused alone, Rho kinase inhibitors (i.e., Rho-associated protein kinaseinhibitors) slightly increased proliferation; and when used alone, aPAK1 inhibitor or a myosin II inhibitor (i.e., non-muscle myosin II (NMII) inhibitor) did not increase proliferation.

In a further experiment, late passage prostate epithelial cells werecultured in KSFM, or KSFM supplemented with A83-01, plus individualcompounds as indicated in FIG. 17, which were tested at variousconcentrations (5 μM, filled bar; 1 μM, checkered bar; and 0.2 μM, openbar). In control experiment with no inhibitor added to KSFM, there wasminimum increase of cell numbers after 5 days. A83-01 significantlyincreased late-passage prostate epithelial cell proliferation, while Rhokinase inhibitor (i.e., Rho-associated protein kinase inhibitor) Y-27632lead to a slight increase of cell proliferation. When both A83-01 andY-27632 were used, they synergistically increased the proliferation ofprostate epithelial cells. Such synergistic effect also was observed forother Rho kinase inhibitors such as SR 3677, GSK 429286 and Thiazovivin.PAK1 inhibitor IPA-3 and myosin II inhibitor (i.e., non-muscle myosin II(NM II) inhibitor) Blebbistatin also synergistically increased prostateepithelial cell proliferation when used together with A83-01 in KSFM. Incontrast, there was little extra increase of prostate epithelial cellproliferation when other ALK5 inhibitors such as GW 788388, SB 431542 orRepSox were used together with A83-01. Thus, several classes ofinhibitors that modulate cytoskeleton integrity synergisticallyincreased late-passage prostate cell proliferation in KSFM, when theywere used together with A83-01. As described above, these include Rhokinase inhibitors (i.e., Rho-associated protein kinase inhibitors) suchas Y-27632, SR 3677, GSK 429286 and Thiazovivin; PAK1 inhibitor IPA-3;and myosin II inhibitor (i.e., non-muscle myosin II (NM II) inhibitor)Blebbistatin.

Example 6 Additional Studies on the Effects of Compounds Including ALK5Inhibitors, Rho Kinase Inhibitors, and Other Compounds on EpithelialCell Proliferation and Other Properties

In this example, proliferation of foreskin keratinocytes, prostateepithelial cells, and bronchial epithelial cells was assessed in thepresence of compounds including ALK5 inhibitors, Rho kinase inhibitors(i.e., Rho-associated protein kinase inhibitors), and other compounds.

Foreskin keratinocytes (FIG. 18), prostate epithelial cells (FIG. 19),and bronchial epithelial cells (FIG. 20) were cultured in either KSFMalone, or KSFM supplemented with 1 μM ALK5 inhibitor A83-01 and 5 μM Rhokinase inhibitor (i.e., Rho-associated protein kinase inhibitor) Y-27632on collagen-coated culture vessels. Population doublings of the cells ineach passage were assessed, and total population doublings were plottedagainst number of days of culture (FIGS. 18, 19 and 20). In KSFM, theepithelial cells showed limited cell replication for only 10 to 20population doublings before ceasing growth. In KSFM with A83-01 andY-27632, the epithelial cells continued to proliferate for 40 to 60additional population doublings. Thus, population doublings of foreskinkeratinocytes, prostate epithelial cells and bronchial epithelial cellsincreased significantly when ALK5 inhibitor A83-01 and Rho kinaseinhibitor (i.e., Rho-associated protein kinase inhibitor) Y-27632 wereused together, which shows that A83-01 and Y-27632 togethersignificantly extend the lifespan of various epithelial cells inculture.

In a further investigation, epithelial cell growth was assessed in thepresence of a beta-adrenergic agonist (i.e., a beta-adrenergic receptoragonist). Human foreskin keratinocytes (HFK) and human bronchialepithelial cells (HBEC) were cultured in KSFM plus 1 μM A83-01 and 5 μMY-27632, supplemented with increasing concentrations of isoproterenol (abeta-adrenergic receptor agonist that increases cytosolic cAMP levels).Cell numbers were counted after six days to calculate cell growthrelative to control. As shown in FIG. 30, isoproterenol furtherincreased epithelial cells growth in KSFM plus A83-01 and Y-27632.

Stable transgenic cell lines were established for various epithelialcells (i.e., human foreskin keratinocytes (HFK), prostate epithelialcells (PrEC), and human bronchial epithelial cells (HBEC)) using alentivirus vector expressing nucleus-localized Red Fluorescence Protein(nRFP). Such transgenic cell lines (e.g., shown in FIG. 24) ubiquitouslyexpress the nRFP reporter gene and are selected through standardantibiotic selection. HFK/nRFP, PrEC/nRFP and HBEC/nRFP cells werecultured for extended periods in KSFM with A83-01 and Y-27632, as shownin FIG. 23.

Karyotypes for various epithelial cells cultured in KSFM plus A83-01 andY-27632 were assessed at early and late passages. Specifically,karyotype analysis was performed on human foreskin keratinocytes (HFK)at passage 3 (13.5 population doublings) and passage 19 (62.0 populationdoublings); human bronchial epithelial cells (HBEC) at passage 4 (11.1population doublings) and passage 16 (45.1 population doublings); andprostate epithelial cells (PrEC) at passage 3 (13.5 populationdoublings) and passage 13 (41.1 population doublings), using metaphasechromosome spreading. The results of the karyotype analysis arepresented in FIG. 21. The cells showed 46 normal chromosomes with nogross karyotypic abnormality after extended culture in the presence ofA83-01 and Y-27632. FIG. 21, lower left panel, shows representativemetaphase chromosome spreads of HFK cells at early passage (p3), andFIG. 21, lower right panel, shows representative metaphase chromosomespreads of HFK cells at late passage (p19).

Average telomere length was assessed for cells cultured in KSFM alone orKSFM plus A83-01 and Y-27632 over several population doublings.Specifically, average telomere length in foreskin keratinocytes culturedin KSFM alone or KSFM plus A83-01 and Y-27632 was determined using aquantitative PCR assay and is represented as a T/S ratio (T, telomere;S, single copy gene) in FIG. 22. The average length of telomeres inforeskin keratinocytes cultured in KSFM plus A83-01 and Y-27632decreased steadily as the population doublings of the culture increased.

Expression of certain genes was assessed at various passages forepithelial cells cultured in KSFM alone or KSFM plus A83-01 and Y-27632.FIG. 27 provides a list of representative genes whose expression levelsare down-regulated or up-regulated in KSFM plus A83-01 and Y-27632.Total RNA was extracted from foreskin keratinocytes cultured in KSFMalone, or KSFM plus A83-01 and Y-27632 at different passages. Geneexpression levels were analyzed by quantitative PCR using RT² Profiler™PCR Array Human Cellular Senescence assay (Qiagen, PAHS-050Z). Severalgenes that are involved in stress response and senescence showedincreased expression when the cells entered senescence at 6th passage inKSFM (i.e., AKT1, ATM, CDKN2A, GADD45A, GLB1, PLAU, SERPINE1 and SOD2),while the expression of these genes was suppressed in KSFM plus A83-01and Y-27632. Likewise, adhesion molecule genes (FN1, THBS1) and anintermediate filament protein (VIM) gene showed increased expressionwhen the cells entered senescence at 6th passage in KSFM, while theexpression of these genes was suppressed in KSFM plus A83-01 andY-27632. The expression of certain genes, CDKN2B, CITED2, CREG1, ID1,MAP2K6, IGFBP3 and IGFBP5 was significantly up-regulated in KSFM plusA83-01 and Y-27632, especially at late passages. Thus, a few genessometimes associated with cellular senescence (such as CDKN2B, CITED2,CREG1, ID1, MAP2K6, IGFBP3 and IGFBP5) showed increased expression inlate passage epithelial cell population cultured in KSFM plus A83-01 andY-27632. Together with the normal karyotype and shorter telomeresobserved in late passage normal epithelial cells cultured in KSFM plusA83-01 and Y-27632, this indicates that normal epithelial cells expandedin KSFM plus A83-01 and Y-27632 gained features such that they aredifferent than the originating epithelial cell population, however theyare not transformed into abnormal cells.

Effects of culture media calcium content on cell behavior were assessedfor epithelial cells cultured in KSFM plus A83-01 and Y-27632. Humanbronchial epithelial cells were grown KSFM (with 90 μM CaCl₂) plusA83-01 and Y-27632. These cells dispersed throughout the culture vessel(FIG. 28, left panel), and few cells formed intercellular connections,even when they were in close proximity to each other. Adding highconcentration (1 mM) of CaCl₂ into KSFM plus A83-01 and Y-27632 causedthe bronchial epithelial cells to aggregate into tight clusters (FIG.28, right panel). Cells in the center of the patches tended to pile up,and boundaries between individual cells generally were not discernable.In certain instances, abnormal elongations formed between clusters.

In a further investigation, effects of culture media calcium content onintercellular junctions was assessed for bronchial epithelial cellscultured in KSFM plus A83-01 and Y-27632. Intercellular junctions wereassessed according to paracellular flow of ions as measured bytrans-epithelium electric resistance (TEER). As shown in FIG. 29 (toppanel), bronchial epithelial cells established tight intercellularjunctions which minimized the paracellular flow of ions, as shown by anincreasing TEER in the presence of high concentration (1 mM) of CaCl₂ inKSFM plus A 83-01 and Y-27632. On contrary, bronchial epithelial cellsfailed to establish tight intercellular junctions under a lowconcentration (90 μM) of CaCl₂ in KSFM plus A 83-01 and Y-27632.Bronchial epithelial cells were plated on porous membrane support(TRANSWELL, Corning, 354474) and maintained for 7 days when the membranewas covered with culture medium (submerged phase, grayed box in FIG. 29,top panel). On day 8, the medium was removed from the apical side of themembrane, and the cells were exposed to air to induce furtherdifferentiation. Further, as shown in FIG. 29 (bottom panel), bronchialepithelial cells established increasing transmembrane electricresistance (TEER) over time in the presence of high concentration (1 mM)of CaCl₂ in KSFM plus A 83-01 and Y-27632. Bronchial epithelial cellswere plated on a porous membrane support (TRANSWELL, Corning, 354474)and maintained for 24 days, with the membrane covered by culture mediumfor the duration of the culture. Trans-epithelium electric resistance(TEER) remained at a high level throughout the culture period. Eachtrace in FIG. 29 (bottom panel) shows a measurement of TEER across oneporous membrane.

Example 7 Identification of Defined Media Compositions for EpithelialCell Proliferation

Bovine pituitary extract is used as a mitogenic supplement in KSFM andin many serum-free cell culture media. In addition to its mitogenicactivity, BPE contains a variety of undefined proteins, lipids andhormones. To identify one or more defined media compositions in whichepithelial cells are capable of proliferating, additional culture mediacompositions were tested. Specifically, epithelial cells wereproliferated in a variety of defined media compositions containing ALK5inhibitors and Rho kinase inhibitors (i.e., Rho-associated proteinkinase inhibitors), and the growth of these cell populations wasassessed. Defined media compositions included one or more componentsselected from M* (MCDB-153 (Modified) Medium (Biological Industries,Cat. No. 01-059-1, which formulation can be found at world wide webaddress bioind.com/page_16682 and in Table 2B below)+epithelial growthfactor (EGF)+acidic fibroblast growth factor (aFGF)+A83-01+Y-27632);fatty-acid free bovine serum albumin (BSA; Sigma, A8806); recombinanthuman serum albumin expressed in Rice (rHA; Sigma, A9731); lipids mix(Chemically Defined Lipid Concentrate; Gibco, 11905-031); and ALBUMAX ILipid-Rich BSA (Gibco, 11020-039). Certain components in MCDB-153 (SigmaAldrich, M7403) and Modified MCDB-153 (Biological Industries, Cat. No.01-059-1) and their concentrations are presented in Tables 2A and 2Bbelow. The lipids mix includes ethyl alcohol and components listed inTable 3 below. ALBUMAX includes BSA and the following fatty acids atbetween about 0.5 to 2.2 mg each/g BSA: Alpha-linolenic acid, Linoleicacid, Oleic acid, Stearic acid, and Palmitic acid.

TABLE 2A MCDB-153 Components Component Concentration (g/L) AmmoniumMetavanadate 0.000000585 Calcium Chloride•Anhydrous 0.00333 CupricSulfate•5H2O 0.00000275 Ferrous Sulfate•7H2O 0.00139 Magnesium Chloride0.05713 Manganese Sulfate 0.000000151 Molybdic Acid•4H2O (ammonium)0.00000124 Nickel Chloride•6H2O 0.00000012 Potassium Chloride 0.11183Sodium Acetate (anhydrous) 0.30153 Sodium Chloride 7.599 SodiumMetasilicate•9H2O 0.000142 Sodium Phosphate Dibasic (anhydrous) 0.284088Sodium Selenite 0.0000038 Stannous Chloride•2H2O 0.000000113 ZincSulfate•7H2O 0.000144 L-Alanine 0.00891 L-Arginine•HCl 0.2107L-Asparagine•H2O 0.015 L-Aspartic Acid 0.00399 L-Cysteine•HCl•H2O0.04204 L-Glutamic Acid 0.01471 L-Glutamine 0.8772 Glycine 0.00751L-Histidine•HCl•H2O 0.01677 L-Isoleucine 0.001968 L-Leucine 0.0656L-Lysine•HCl 0.01827 L-Methionine 0.00448 L-Phenylalanine 0.00496L-Proline 0.03453 L-Serine 0.06306 L-Threonine 0.01191 L-Tryptophan0.00306 L-Tyrosine•2Na 0.00341 L-Valine 0.03513 D-Biotin 0.0000146Choline Chloride 0.01396 Folic Acid 0.00079 myo-Inositol 0.01802Niacinamide 0.00003663 D-Pantothenic Acid (hemicalcium) 0.000238Pyridoxine•HCl 0.00006171 Riboflavin 0.0000376 Thiamine•HCl 0.000337Vitamin B-12 0.000407 Adenine•HCl 0.03088 D-Glucose 1.081 HEPES 6.6Phenol Red•Na 0.001242 Putrescine•2HCl 0.000161 Pyruvic Acid•Na 0.055Thioctic Acid 0.000206 Thymidine 0.000727

TABLE 2B Modified MCDB-153 Components Component Concentration (g/L*)Ammonium Metavanadate 0.000000585 Calcium Chloride•Anhydrous 0.00333Cupric Sulfate•5H2O 0.00000275 Ferrous Sulfate•7H2O 0.00139 MagnesiumChloride 0.05713 Molybdic Acid•4H2O (ammonium) 0.00000124 NickelChloride•6H2O 0.00000012 Potassium Chloride 0.11183 Sodium Acetate(anhydrous) 0.30153 Sodium Chloride 7.599 Sodium Metasilicate•9H2O0.000142 Sodium Phosphate Dibasic (anhydrous) 0.284088 Sodium Selenite0.0000038 Stannous Chloride•2H2O 0.000000113 Zinc Sulfate•7H2O 0.000144L-Alanine 0.0178 L-Arginine•HCl 0.2107 L-Asparagine•H2O 0.030 L-AsparticAcid 0.01729 L-Cystine•HCl•H2O 0.04204 L-Glutamic acid 0.0294L-Glutamine 0.8772 Glycine 0.0150 L-Histidine•HCl•H2O 0.01677L-Isoleucine 0.001968 L-Leucine 0.0656 L-Lysine•HCl 0.01827 L-Methionine0.00448 L-Phenylalanine 0.00496 L-Proline 0.04603 L-Serine 0.07356L-Threonine 0.01191 L-Tryptophan 0.00306 L-Tyrosine•2Na 0.00341 L-Valine0.03513 D-Biotin 0.0000146 Choline Chloride 0.01396 Folic Acid 0.00079myo-Inositol 0.01802 Niacinamide 0.00003663 D-Pantothenic Acid(hemicalcium) 0.000238 Pyridoxine•HCl 0.00006171 Riboflavin 0.0000376Thiamine•HCl 0.000337 Vitamin B-12 0.000407 Adenine•HCl 0.03088D-Glucose 1.081 HEPES 6.6 Phenol Red•Na 0.001242 Putrescine•2HCl0.000161 Pyruvic Acid•Na 0.055 Thioctic Acid 0.000206 Thymidine 0.000727Hydrocortisone 200 nM Triiodothyronine 10 nM Testosteron 10 nM Insulin5.0 mg/L Transferrin (Iron-free) 5.0 mg/L Sodium selenite 5.0 μg/L*Concentration is in g/L except where noted otherwise.

TABLE 3 Lipids Mix Components Component Concentration (mg/L) ArachidonicAcid 2.0 Cholesterol 220.0 DL-alpha-Tocopherol Acetate 70.0 LinoleicAcid 10.0 Linolenic Acid 10.0 Myristic Acid 10.0 Oleic Acid 10.0Palmitic Acid 10.0 Palmitoleic Acid 10.0 Pluronic F-68 90000.0 StearicAcid 10.0 Tween 80 ® (polysorbate 80) 2200.0

HFK cells and HBEC cells were grown in M*; M*+ALBUMAX (at 1000 μg/mL,500 μg/mL, 250 μg/mL, 125 μg/mL, 62.5 μg/mL, 31.3 μg/mL and 15.6 μg/mL);M*+BSA+lipids mix (at 1:50 dilution, 1:100 dilution, 1:200 dilution,1:400 dilution, 1:800 dilution, 1:1600 dilution, and 1:3200 dilution);or M*+rHA+lipids mix (at 1:50 dilution, 1:100 dilution, 1:200 dilution,1:400 dilution, 1:800 dilution, 1:1600 dilution, and 1:3200 dilution).Growth of the cell populations was assessed and the results arepresented as fold increase of cell number in FIG. 25 for HFK cells andFIG. 26 for HBEC cells. The results show that albumin and a lipidsmixture can be used to support epithelial cell proliferation without theneed for bovine pituitary extract (BPE) supplementation.

Example 8 Epithelial Cell Gene Expression Profiles

In this example, gene expression profiles are described for epithelialcells grown in various serum-free media conditions.

Total RNAs were extracted from human foreskin keratinocytes cultured inKSFM at passages 2 and 6, or KSFM plus A 83-01 (1 μM) and Y-27632 (5 μM)at passages 2, 13 and 23; and airway epithelial cells cultured in KSFMplus A 83-01 (1 μM) and Y-27632 (5 μM) at passages 2 and 8. The cellculture media for the airway epithelial cells also includedisoproterenol (3 μM). Gene expression levels were analyzed byquantitative RT-PCR using customized RT² Profiler™ PCR Array (Qiagen).Total RNAs from human small intestine or lung tissues (Clontech) wereincluded as controls. Gene expression levels relative to that of Actin Bwere calculated using 2̂(Ct_(actinB)−Ct_(gene)), where Ct_(actinB) orCt_(gene) is the number of cycles required for the fluorescent signal ofquantitative PCR reaction to cross a defined threshold. Ct_(actinB) wasgenerally around 18. The expression level of a gene was considerednon-detectable (ND) if the Ct_(gene) was higher than 35. The expressionlevel of a gene was considered low if the Ct_(gene) was less than 35 andgreater than or equal to 30. The expression level of a gene wasconsidered medium or moderate if the Ct_(gene) was less than 29 andgreater than or equal to 22. The expression level of a gene wasconsidered high if the Ct_(gene) was less than 22.

As shown in Table 4 below, epithelial cells grown in KSFM plus A 83-01and Y-27632 expressed high levels of genes that typically are expressedin basal epithelial cells (ITGA6, ITGB4, KRT14, KRT15, KRT5 and TP63).These cells also lacked expression of certain pluripotent stem cellmarkers such as LIN28A, NANOG, POU5F1/OCT4 and SOX2, and they expresseda moderate level of KLF4. The cells also did not express or expressedvery low levels of genes that typically are expressed in terminallydifferentiated epithelial cells, including CFTR, FOXJ1, IVL, KRT1,KRT10, KRT20, LOR, MUC1, MUC5AC, SCGB1A1, SFTPB and SFTPD. None of thegenes highly expressed in gastric, intestinal, or pancreatic epithelialcells were detected in the cells grown in KSFM plus A 83-01 and Y-27632,including CD34, HNF1A, HNF4A, IHH, KIT, LGR5, PDX1, and PROM1/CD133.

TABLE 4 Gene expression profile of epithelial cells grown in KSFM plusA83-01 and Y-27632 Gene Small Name GenBank Description Intestine LungGenes that are enriched in basal epithelial cells ITGA6 NM_000210Integrin, Alpha 6 0.032 0.015 ITGB4 NM_000213 Integrin, Beta 4 0.00410.0019 KRT14 NM_000526 Keratin 14, Type I 0.00016 0.00021 KRT15NM_002275 Keratin 15, Type I 0.0002 0.0015 KRT5 NM_000424 Keratin 5,Type II 0.00013 0.0028 TP63 NM_003722 Tumor Protein P63 ND 0.00057Markers for pluripotent stem cells KLF4 NM_004235 Kruppel-Like Factor 40.022 0.038 LIN28A NM_024674 Lin-28 Homolog A 0.00085 0.00031 NANOGNM_024865 Nanog Homeobox 0.0013 0.00057 POU5F1 NM_002701 POU Class 5Homeobox 1 0.0011 0.00066 SOX2 NM_003106 SRY (Sex Determining RegionY)-Box 2 0.00036 0.00021 Genes that are enriched in airway epithelialcells BMP7 NM_001719 Bone Morphogenetic Protein 7 0.00058 0.00045 HEY2NM_012259 Hes-Related Family BHLH 0.0022 0.0022 Transcription FactorWith YRPW Motif 2 NGFR NM_002507 Nerve Growth Factor Receptor 0.000840.00011 Gene that is enriched in keratinocytes ZFP42 NM_174900 ZFP42Zinc Finger Protein ND ND Genes that make up keratin intermediatefilaments KRT1 NM_006121 Keratin 1, Type II 0.00029 0.00011 KRT10NM_000421 Keratin 10, Type I ND ND KRT14 NM_000526 Keratin 14, Type I0.00015 0.00021 KRT15 NM_002275 Keratin 15, Type I 0.0002 0.0015 KRT16NM_005557 Keratin 16, Type I ND 0.00016 KRT18 NM_000224 Keratin 18, TypeI 0.058 0.015 KRT19 NM_002276 Keratin 19, Type I 0.13 0.043 KRT20NM_019010 Keratin 20, Type I 0.078 ND KRT4 NM_002272 Keratin 4, Type II0.00029 0.00076 KRT5 NM_000424 Keratin 5, Type II 0.00012 0.0027 KRT6ANM_005554 Keratin 6A, Type II 0.00015 0.00016 KRT7 NM_005556 Keratin 7,Type II ND 0.023 KRT8 NM_002273 Keratin 8, Type II 0.0080 0.0017 Genesthat are expressed in terminally differentiated cells CFTR NM_000492Cystic Fibrosis Transmembrane 0.0013 0.00069 Conductance Regulator FOXJ1NM_001454 Forkhead Box J1 ND 0.0043 IVL NM_005547 Involucrin ND ND KRT1NM_006121 Keratin 1, Type II 0.00029 0.00011 KRT10 NM_000421 Keratin 10,Type I ND ND KRT20 NM_019010 Keratin 20, Type I 0.078 ND LOR NM_000427Loricrin ND ND MUC1 NM_001018016 Mucin 1, Cell Surface Associated0.00066 0.018 MUC5AC XM_003403450 Mucin 5AC, Oligomeric Mucus/Gel- ND NDForming SCGB1A1 NM_003357 Secretoglobin, Family 1A, Member 1 ND 0.18SFTPB NM_000542 Surfactant Protein B ND 0.082 SFTPD NM_003019 SurfactantProtein D 0.00017 0.038 Markers for gastric/intestinal/pancreaticepithelium stem cells AXIN2 NM_004655 Axin 2 0.0050 0.0029 BMP4NM_130851 Bone Morphogenetic Protein 4 0.0031 0.0017 BMP5 NM_021073 BoneMorphogenetic Protein 5 0.0043 0.017 BMP6 NM_001718 Bone MorphogeneticProtein 6 0.0019 0.0034 CD34 NM_001773 CD34 Molecule 0.035 0.085 CFTRNM_000492 Cystic Fibrosis Transmembrane 0.0013 0.00069 ConductanceRegulator DLL4 NM_019074 Delta-Like 4 0.00039 0.0029 HNF1A NM_000545HNF1 Homeobox A 0.0046 ND HNF4A NM_178849 Hepatocyte Nuclear Factor 4,Alpha 0.0055 ND IHH NM_002181 Indian Hedgehog 0.00025 ND KIT NM_000222V-Kit Hardy-Zuckerman 4 Feline 0.0021 0.0026 Sarcoma Viral OncogeneHomolog KRT20 NM_019010 Keratin 20, Type I 0.078 ND LGR5 NM_003667Leucine-Rich Repeat Containing G 0.0017 0.00073 Protein-Coupled Receptor5 PDX1 NM_000209 Pancreatic And Duodenal Homeobox 1 0.0082 ND PROM1NM_006017 Prominin 1 0.0039 0.0017 Foreskin Keratinocyte AirwayEpithelial Cells KSFM + KSFM + KSFM + KSFM + KSFM + Gene KSFM KSFM A + YA + Y A + Y A + Y A + Y Name (p2) (p6) (p2) (p13) (p23) (p2) (p8) Genesthat are enriched in basal epithelial cells ITGA6 0.25 0.27 0.17 0.210.39 0.22 0.21 ITGB4 0.039 0.047 0.031 0.027 0.018 0.034 0.026 KRT141.45 0.85 2.53 2.19 2.63 1.19 1.21 KRT15 0.058 0.053 0.35 0.086 1.030.25 0.13 KRT5 0.73 0.91 1.52 1.53 1.66 1.47 1.03 TP63 0.046 0.043 0.0920.047 0.033 0.066 0.03 Markers for pluripotent stem cells KLF4 0.0140.011 0.044 0.028 0.036 0.024 0.012 LIN28A ND ND 0.00012 ND 0.00016 NDND NANOG ND 0.00031 ND ND 0.00013 0.00044 ND POU5F1 0.00046 0.000910.00040 0.00051 0.0015 0.00036 0.00039 SOX2 ND ND ND ND ND 0.00260.00097 Genes that are enriched in airway epithelial cells BMP7 ND ND ND0.00023 0.00095 0.0041 0.0041 HEY2 0.00025 ND 0.00081 ND ND 0.24 0.074NGFR 0.00019 0.00011 0.0018 0.00095 0.000091 0.0046 0.0022 Gene that isenriched in keratinocytes ZFP42 0.00096 0.0012 0.00077 0.0013 0.0022 NDND Genes that make up keratin intermediate filaments KRT1 ND 0.00014 ND0.00058 0.0015 ND ND KRT10 ND ND 0.00024 0.00078 0.020 0.00051 ND KRT141.45 0.84 2.53 2.19 2.62 1.19 1.21 KRT15 0.057 0.053 0.35 0.085 1.030.25 0.13 KRT16 0.0045 0.032 0.088 0.038 0.050 0.024 0.015 KRT18 0.0750.063 0.13 0.043 0.024 0.048 0.025 KRT19 0.16 0.39 0.077 0.33 0.81 0.620.41 KRT20 ND ND ND ND ND ND ND KRT4 ND 0.00015 0.00013 0.0048 0.400.0039 0.0098 KRT5 0.72 0.91 1.51 1.52 1.66 1.47 1.032 KRT6A 0.54 1.340.75 0.87 2.87 0.85 0.75 KRT7 0.041 0.11 0.0049 0.00014 0.0011 0.0230.045 KRT8 0.0011 0.0011 0.0010 0.00097 0.00023 0.0010 0.00023 Genesthat are expressed in terminally differentiated cells CFTR ND ND ND NDND ND ND FOXJ1 ND ND ND ND ND ND ND IVL 0.00066 0.022 0.00085 0.00270.19 0.00039 0.0023 KRT1 ND 0.00014 ND 0.00058 0.0015 ND ND KRT10 ND ND0.00024 0.00078 0.020 0.00051 0.000083 KRT20 ND ND ND ND ND ND ND LOR NDND ND ND 0.00016 ND ND MUC1 ND 0.00047 0.00034 0.00022 0.0030 0.000210.00023 MUC5AC ND ND ND ND ND ND ND SCGB1A1 ND ND ND ND ND ND ND SFTPBND ND ND ND ND ND ND SFTPD ND ND ND ND 0.00027 ND ND Markers forgastric/intestinal/pancreatic epithelium stem cells AXIN2 ND ND ND ND NDND ND BMP4 0.00014 0.00014 ND 0.00022 0.00048 0.00035 0.00032 BMP5 ND NDND ND ND ND ND BMP6 ND 0.00011 ND ND ND ND ND CD34 ND ND 0.00010 ND NDND ND CFTR ND ND ND ND ND ND ND DLL4 ND ND ND ND ND ND ND HNF1A ND ND NDND ND ND ND HNF4A ND ND ND ND ND ND ND IHH ND ND ND ND ND ND ND KIT NDND ND ND ND ND ND KRT20 ND ND ND ND ND ND ND LGR5 ND ND ND ND ND ND NDPDX1 ND ND ND ND ND ND ND PROM1 ND ND ND ND ND ND ND

Example 9 Characterization of Epithelial Cells in Culture

In this example, certain characteristics are described for epithelialcells grown in various feeder-free and serum-free media conditions.

Differentiation of Bronchial Epithelial Cells into Bronchospheres

Passage 2 human bronchial epithelial cells cultured in KSFM supplementedwith 1 μM A 83-01 and 5 μM Y-27632 (KSFM+A+Y) were removed from KSFM+A+Yconditions and embedded in Matrigel® as single cells, and cultured inClonetics™ B-ALI™ air-liquid interface medium (high calciumdifferentiation medium, Lonza) for 14 days. FIG. 31 shows bronchialepithelial cells differentiated into bronchospheres. The top panel ofFIG. 31 shows cells viewed at lower (4×) magnification, and the bottompanel of FIG. 31 shows cells viewed at higher (20×) magnification. Largebronchospheres with visible lumen are shown in the bottom panel of FIG.31.

Characterization of Epithelial Cells after Exposure to High CalciumConcentrations

Dome-like structures formed in keratinocyte and bronchial epithelialcell cultures in the presence of high concentration of CaCl₂.Specifically, late passage human bronchial epithelial cells (HBEC) andlate passage human foreskin keratinocytes (HFK) cultured in KSFMsupplemented with 1 μM A 83-01 and 5 μM Y-27632 (KSFM+A+Y) at low CaCl₂(90 μM) were allowed to reach confluence in 6-well plates. The cellsremained in the KSFM+A+Y conditions and the CaCl₂ concentration wasraised to 1.5 mM to induce differentiation of the epithelial cells. Manydome-like structures were formed after 7 to 10 days and are shown inFIG. 32A to FIG. 32D.

Tight junction formation was observed between keratinocytes afterexposure to a high concentration of CaCl₂. Specifically, late passagehuman foreskin keratinocytes (HFK) cultured in KSFM supplemented with 1μM A83-01 and 5 μM Y-27632 (KSFM+A+Y) at low CaCl₂ (90 μM) were allowedto reach confluence. The cells remained in the KSFM+A+Y conditions andthe CaCl₂ concentration was raised to 1.5 mM to induce differentiation.The presence of intercellular tight junctions was revealed byimmunofluorescence staining of tight junction protein ZO-1 using amonoclonal antibody conjugated to Alexa Fluor® 488 (ThermoFisher,339188), and is shown in FIG. 33.

Keratinocytes established increasing transmembrane electric resistance(TEER) over time in air-liquid-interface differentiation, in thepresence of high concentration (1.5 mM) of CaCl₂ in KSFM plus A 83-01and Y-27632. Specifically, human foreskin keratinocytes (HFK) previouslycultured in KSFM supplemented with 1 μM A83-01 and 5 μM Y-27632(KSFM+A+Y) at low CaCl₂ (90 μM) were plated on a porous membrane support(TRANSWELL, Corning, 354474) and maintained for 14 days. The cells werecovered by culture medium (KSFM+A+Y and 1.5 mM of CaCl₂) for the firstday (submerged phase, grayed box in FIG. 34) and exposed to air for theremaining days. As shown in FIG. 34, trans-epithelium electricresistance (TEER) reached very high levels throughout the cultureperiod.

Keratinocytes formed an epidermal-like structure over time inair-liquid-interface differentiation, in the presence of highconcentration (1.5 mM) of CaCl₂ in KSFM plus A 83-01 and Y-27632.Specifically, human foreskin keratinocytes (HFK) previously cultured inKSFM supplemented with 1 μM A83-01 and 5 μM Y-27632 (KSFM+A+Y) at lowCaCl₂ (90 μM) were plated on a porous membrane support (TRANSWELL,Corning, 354474) and maintained for 14 days. The cells were covered byculture medium (KSFM+A+Y and 1.5 mM of CaCl₂) for the first day andexposed to air for the remaining days. At the end of experiment (i.e.,on day 14), the culture was fixed in 4% paraformaldehyde, embedded inparaffin and sectioned for haematoxylin and eosin staining (H&E) toreveal its structure. As shown in FIG. 35, the cells had differentiatedinto multi-layer structures, with layers resembling stratum corneum,stratum granulosm, stratum spinosum, and stratum basale.

Further Characterization of Epithelial Cell Culture

Single cell cloning and expansion of keratinocytes was examined.Specifically, a single human foreskin keratinocyte (HFK) at late passage(previously cultured in KSFM plus 1 μM A 83-01, 5 μM Y-27632 and 3 μMisoproterenol) was plated onto a collagen I coated 384-well plate andcultured in KSFM plus 1 μM A 83-01, 5 μM Y-27632 and 3 μM isoproterenol.Over 10 days, the cell divided into more than 1000 cells and formed acolony, as shown in FIG. 36.

Heterogeneity in cellular morphology was observed for keratinocyteprogeny derived from a single cell. Specifically, the single-cellderived colony described above and shown in FIG. 36 was further expandedin a T-25 flask in KSFM plus 1 μM A 83-01, 5 μM Y-27632 and 3 μMisoproterenol.

Heterogeneity in cellular morphology (e.g., cell size) was observed andis shown in FIG. 37. Mitotic cells were identified by a characteristicrounded morphology, a phase bright halo, and a central dark band(indicating condensed chromosomes).

Single cell colony forming efficiency was examined for certainepithelial cell types cultured in KSFM plus A 83-01, Y-27632 andisoproterenol. Specifically, late passage human foreskin keratinocytes(HFK) and human bronchial epithelial cells (HBEC) previously cultured inKSFM plus 1 μM A 83-01, 5 μM Y-27632 and 3 μM isoproterenol were seededonto collagen coated 384-well plates at one cell per well and allowed togrow for 10 days in KSFM plus 1 μM A 83-01, 5 μM Y-27632 and 3 μMisoproterenol. On day 10, the number of cells in each well wasdetermined. The number of wells (i.e., colonies) having less than 20cells, having 21-100 cells, having 101-500 cells, or having more than500 cells were tallied and plotted, and the results are presented inFIG. 38.

Expansion of Epithelial Cells Cultured in Different Media Conditions

Epithelial cells from different tissues were cultured to evaluate theirpotential for expansion in different culture media. The cells wereobtained from ThermoFisher/Gibco (HFKn and HEKa) or Lonza (HMEC, PrEC,HBEC, SAEC and DHBE-CF). Fold expansion was calculated using the formulaF=2, where F=the fold of expansion after n population doublings, and theresults are presented in Table 5 below.

TABLE 5 Fold expansion of epithelial cells Donor Population Cell TypeAge Medium Fold expansion doublings HFKn neonatal KSFM 122,295 16.9 HFKnneonatal KSFM + A + Y (*) 3,115,599,965,857,640,000,000 71.4 HFKnneonatal KSFM + A + IPA 3,333,095,978,582 41.6 HFKn neonatal KSFM + A +B 1,744,298,739 30.7 HFKn neonatal KSFM + A + GSK 315,751,799,532 38.2HEKa adult KSFM 2,896 11.5 HEKa adult KSFM + A + Y67,232,112,528,152,800 55.9 HEKa adult KSFM + A + B 416,636,997,323 38.6HEKa adult KSFM + A + GSK 90,675,893,177 36.4 HMEC adult KSFM 5 2.3 HMECadult KSFM + A + Y 15,314,887,470,577 43.8 PrEC adult KSFM 489,178 18.9PrEC adult PrGM 4,390 12.1 PrEC adult KSFM + A 10,327,588 23.3 PrECadult KSFM + Y 3,178,688 21.6 PrEC adult KSFM + A + Y 7,657,443,735,28842.8 PrEC adult KSFM + A + IPA 9,206,463,941 33.1 HBEC adult KSFM 2,35311.2 HBEC adult KSFM + A 1,123,836 20.1 HBEC adult KSFM + Y 561,918 19.1HBEC adult KSFM + A + Y 228,628,724,347,545 47.7 HBEC adult KSFM + A +IPA 15,314,887,470,577 43.8 SAEC adult KSFM 21 4.4 SAEC adult KSFM + A +Y (*) 38,543,921 25.2 DHBE- adult KSFM + A + Y 5,173,277,483,525,74052.2 CF (cystic fibrosis)

HFKn, neonatal human foreskin keratinocyte. HEKa, adult human epidermalkeratinocyte. HMEC, human mammary epithelial cells (female). PrEC, humanprostate epithelial cells. HBEC, human bronchial epithelial cells. SAEC,human small airway epithelial cells. DHBE-CF, diseased human bronchialepithelial cells from cystic fibrosis patient. * notes that the cellculture was voluntarily suspended after it achieved much more folds ofexpansion than in KSFM, the population was still undergoing activedivisions when the experiment was suspended.

KSFM, Keratinocyte-SFM (Gibco/Thermo Fisher). PrGM, Prostate EpithelialCell Growth Medium (Lonza). A, ALK5 inhibitor A83-01. Y, Rho kinaseinhibitor (i.e., Rho-associated protein kinase inhibitor) Y-27632. B,myosin II inhibitor blebbistatin. IPA, Group I p21-activated kinase(PAK1) inhibitor. GSK, Rho kinase inhibitor (i.e., Rho-associatedprotein kinase inhibitor) GSK-429286.

Example 10 Examples of Embodiments

The examples set forth below illustrate certain embodiments and do notlimit the technology.

A1. A method for proliferating differentiated epithelial cells ex vivo,which method comprises:

-   -   a) culturing differentiated epithelial cells under serum-free        and feeder-cell free conditions; and    -   b) inhibiting TGF-beta signaling in the differentiated        epithelial cells during the culturing in (a).

A1.1 A method for proliferating formerly quiescent epithelial cells exvivo, which method comprises:

-   -   a) culturing formerly quiescent epithelial cells under        serum-free and feeder-cell free conditions; and    -   b) inhibiting TGF-beta signaling in the formerly quiescent        epithelial cells during the culturing in (a).

A1.2 A method for proliferating lineage-committed epithelial cells exvivo, which method comprises:

-   -   a) culturing lineage-committed epithelial cells under serum-free        and feeder-cell free conditions; and    -   b) inhibiting TGF-beta signaling in the lineage-committed        epithelial cells during the culturing in (a).

A2. A method for proliferating epithelial cells ex vivo, which methodcomprises:

-   -   a) culturing epithelial cells under feeder-cell free conditions;    -   b) inhibiting TGF-beta signaling in the epithelial cells during        the culturing in (a); and    -   c) inhibiting the activity of p21-activated kinase (PAK) in the        epithelial cells during the culturing in (a).

A2.1 The method of embodiment A2, wherein the epithelial cells comprisedifferentiated epithelial cells.

A2.2 The method of embodiment A2, wherein the epithelial cells compriseformerly quiescent epithelial cells.

A2.3 The method of embodiment A2, wherein the epithelial cells compriselineage-committed epithelial cells.

A3. A method for proliferating epithelial cells ex vivo, which methodcomprises:

-   -   a) culturing epithelial cells under serum-free and feeder-cell        free conditions;    -   b) inhibiting TGF-beta signaling in the epithelial cells during        the culturing in (a); and    -   c) inhibiting the activity of myosin II in the epithelial cells        during the culturing in (a).

A3.1 The method of embodiment A3, wherein the epithelial cells comprisedifferentiated epithelial cells.

A3.2 The method of embodiment A3, wherein the epithelial cells compriseformerly quiescent epithelial cells.

A3.3 The method of embodiment A3, wherein the epithelial cells compriselineage-committed epithelial cells.

A3.4 The method of any one of embodiments A3 to A3.3, wherein the myosinII is a non-muscle myosin II (NM II).

A4. The method of any one of embodiments A2 to A3.4, wherein theculturing in (a) is performed in the presence of a serum containingmedium.

A4.1 The method of any one of embodiments A2 to A3.4, wherein theculturing in (a) is performed in the presence of a serum-free medium.

A5. A method for proliferating differentiated epithelial cells ex vivo,which method comprises:

-   -   a) culturing differentiated epithelial cells under feeder-cell        free conditions;    -   b) activating telomerase reverse transcriptase in the        differentiated epithelial cells; and    -   c) modulating cytoskeletal structure in the differentiated        epithelial cells.

A5.1 A method for proliferating formerly quiescent epithelial cells exvivo, which method comprises:

-   -   a) culturing formerly quiescent epithelial cells under        feeder-cell free conditions;    -   b) activating telomerase reverse transcriptase in the formerly        quiescent epithelial cells; and    -   c) modulating cytoskeletal structure in the formerly quiescent        epithelial cells.

A5.2 A method for proliferating lineage-committed epithelial cells exvivo, which method comprises:

-   -   a) culturing lineage-committed epithelial cells under        feeder-cell free conditions;    -   b) activating telomerase reverse transcriptase in the        lineage-committed epithelial cells; and    -   c) modulating cytoskeletal structure in the lineage-committed        epithelial cells.

A5.3 The method of embodiment A5, A5.1 or A5.2, wherein TGF-betasignaling is inhibited in (b).

A5.4 The method of any one of embodiments A1 to A5.3, wherein (a) and(b) are performed at the same time; or wherein (a), (b) and (c) areperformed at the same time.

A5.5 The method of any one of embodiments A1 to A5.4, wherein theepithelial cells are frozen and thawed prior to (a).

A6. The method of any one of embodiments A1 to A5.5, wherein theactivity of one or more TGF-beta receptors is inhibited in (b).

A7. The method of embodiment A6, wherein one or more TGF-betareceptor-ligand interactions are inhibited in (b).

A8. The method of embodiment A6 or A7, wherein the one or more TGF-betareceptors comprise a TGF-beta type I receptor.

A9. The method of embodiment A8, wherein the TGF-beta type I receptor isselected from ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, ALK7 and ALK8.

A10. The method of embodiment A8, wherein the one or more TGF-betareceptors comprise ALK5.

A11. The method of any one of embodiments A1 to A10, wherein inhibitingTGF-beta signaling comprises use of one or more TGF-beta inhibitorsand/or one or more TGF-beta signaling inhibitors.

A12. The method of embodiment A11, wherein the one or more TGF-betainhibitors and/or the one or more TGF-beta signaling inhibitors bind toone or more TGF-beta receptors or one or more TGF-beta ligands or both.

A13. The method embodiment A11 or A12, wherein the one or more TGF-betainhibitors and/or the one or more TGF-beta signaling inhibitors disruptone or more TGF-beta receptor-ligand interactions.

A14. The method of embodiment A11, A12 or A13, wherein the one or moreTGF-beta inhibitors and/or the one or more TGF-beta signaling inhibitorsdo not comprise a recombinant protein.

A15. The method of any one of embodiments A11 to A14, wherein the one ormore TGF-beta inhibitors and/or the one or more TGF-beta signalinginhibitors do not comprise Noggin, DAN, Cerberus or Gremlin.

A16. The method of any one of embodiments A11 to A15, wherein the one ormore TGF-beta inhibitors and/or the one or more TGF-beta signalinginhibitors comprise one or more ALK5 inhibitors.

A17. The method of embodiment A16, wherein the one or more ALK5inhibitors comprise one or more small molecule ALK5 inhibitors.

A18. The method of embodiment A17, wherein the one or more ALK5inhibitors comprise one or more ATP analogs.

A19. The method of any one of embodiments A16 to A18, wherein at leastone of the one or more ALK5 inhibitors comprises the structure ofFormula A:

wherein:

-   -   X, Y and Z independently are chosen from N, C and O;    -   R¹, R² and R³ independently are chosen from hydrogen, C1-C10        alkyl, substituted C1-C10 alkyl, C3-C9 cycloalkyl, substituted        C3-C9 cycloalkyl, C5-C10 aryl, substituted C5-C10 aryl, C5-C10        cycloaryl, substituted C5-C10 cycloaryl, C5-C9 heterocyclic,        substituted C5-C9 heterocyclic, C5-C9 hetercycloaryl,        substituted C5-C9 heterocycloaryl, -linker-(C3-C9 cycloalkyl),        -linker-(substituted C3-C9 cycloalkyl), -linker-(C5-C10 aryl),        -linker-(substituted C5-C10 aryl), -linker-(C5-C10 cycloaryl),        -linker-(substituted C5-C10 cycloaryl), -linker-(C5-C9        heterocyclic), -linker-(substituted C5-C9 heterocyclic),        -linker-(C5-C9 hetercycloaryl), -linker-(substituted C5-C9        heterocycloaryl);    -   n is 0 or 1;    -   R⁴, R⁵ and R⁶ independently are chosen from hydrogen, C1-C10        alkyl, substituted C1-C10 alkyl, C1-C10 alkoxy, substituted        C1-C10 alkoxy, C1-C6 alkanoyl, C1-C6 alkoxycarbonyl, substituted        C1-C6 alkanoyl, substituted C1-C6 alkoxycarbonyl, C3-C9        cycloalkyl, substituted C3-C9 cycloalkyl, C5-C10 aryl,        substituted C5-C10 aryl, C5-C10 cycloaryl, substituted C5-C10        cycloaryl, C5-C9 heterocyclic, substituted C5-C9 heterocyclic,        C5-C9 hetercycloaryl, substituted C5-C9 heterocycloaryl,        -linker-(C3-C9 cycloalkyl), -linker-(substituted C3-C9        cycloalkyl), -linker-(C5-C10 aryl), -linker-(substituted C5-C10        aryl), -linker-(C5-C10 cycloaryl), -linker-(substituted C5-C10        cycloaryl), -linker-(C5-C9 heterocyclic), -linker-(substituted        C5-C9 heterocyclic), -linker-(C5-C9 hetercycloaryl),        -linker-(substituted C5-C9 heterocycloaryl); and    -   the substituents on the substituted alkyl, alkoxy, alkanoyl,        alkoxycarbonyl cycloalkyl, aryl, cycloaryl, heterocyclic or        heterocycloaryl groups are hydroxyl, C1-C10 alkyl, hydroxyl        C1-C10 alkylene, C1-C6 alkoxy, C3-C9 cycloalkyl, C5-C9        heterocyclic, C1-6 alkoxy C1-6 alkenyl, amino, cyano, halogen or        aryl.

A20. The method of any one of embodiments A16 to A19, wherein the one ormore ALK5 inhibitors are selected from A83-01, GW788388, RepSox, and SB431542.

A21. The method of embodiment A20, wherein the one or more ALK5inhibitors comprise A83-01.

A22. The method of any one of embodiments A16 to A21, wherein the one ormore ALK5 inhibitors binds to ALK5 or one or more ALK5 ligands or both.

A23. The method of any one of embodiments A16 to A22, wherein the one ormore ALK5 inhibitors disrupt one or more ALK5-ligand interactions.

A24. The method of any one of embodiments A1 to A23, wherein the methodcomprises activating telomerase reverse transcriptase the epithelialcells.

A24.1 The method of any one of embodiments A1 to A24, wherein the methodcomprises modulating cytoskeletal structure in the epithelial cells.

A24.2 The method of any one of embodiments A1 to A24.1, wherein themethod comprises:

-   -   a) activating telomerase reverse transcriptase the epithelial        cells; and    -   b) modulating cytoskeletal structure in the epithelial cells.

A25. The method of any one of embodiments A1 to A24.2, furthercomprising inhibiting the activity of Rho kinase and/or Rho-associatedprotein kinase in the epithelial cells during the culturing in (a).

A26. The method of embodiment A25, wherein Rho kinase and/orRho-associated protein kinase is selected from Rho kinase 1 (ROCK 1) andRho kinase 2 (ROCK 2).

A27. The method of embodiment A25 or A26, wherein inhibiting theactivity of Rho kinase and/or Rho-associated protein kinase comprisesuse of one or more Rho kinase inhibitors and/or one or moreRho-associated protein kinase inhibitors.

A28. The method of embodiment A27, wherein the one or more Rho kinaseinhibitors and/or the one or more Rho-associated protein kinaseinhibitors comprise one or more small molecule Rho kinase inhibitorsand/or one or more small molecule Rho-associated protein kinaseinhibitors.

A29. The method of embodiment A28, wherein the one or more Rho kinaseinhibitors and/or the one or more Rho-associated protein kinaseinhibitors is selected from Y-27632, SR 3677, thiazovivin, HA1100hydrochloride, HA1077 and GSK-429286.

A30. The method of embodiment A29, wherein the one or more Rho kinaseinhibitors and/or the one or more Rho-associated protein kinaseinhibitors comprise Y-27632.

A30.1 The method of any one of embodiments A1 to A24, which does notcomprise inhibiting the activity of Rho kinase and/or Rho-associatedprotein kinase in the epithelial cells during the culturing in (a).

A31. The method of any one of embodiments A1 to A30.1, furthercomprising inhibiting the activity of p21-activated kinase (PAK) in theepithelial cells during the culturing in (a).

A32. The method of embodiment A31, wherein the PAK is selected fromPAK1, PAK2, PAK3 and PAK4.

A33. The method of embodiment A32, wherein the PAK is PAK1.

A34. The method of embodiment A33, wherein inhibiting the activity ofPAK1 comprises use of one or more PAK1 inhibitors.

A35. The method of embodiment A34, wherein the one or more PAK1inhibitors comprise one or more small molecule PAK1 inhibitors.

A36. The method of embodiment A35, wherein the one or more PAK1inhibitors comprise IPA3.

A37. The method of any one of embodiments A1 to A36, further comprisinginhibiting the activity of myosin II in the epithelial cells during theculturing in (a).

A37.1 The method of embodiment A37, wherein the myosin II is anon-muscle myosin II (NM II).

A37.2 The method of embodiment A37 or A37.1, wherein inhibiting theactivity of myosin II comprises use of one or more myosin II inhibitors.

A37.3 The method of embodiment A37.2, wherein the one or more myosin IIinhibitors comprise one or more small molecule myosin II inhibitors.

A37.4. The method of embodiment A37.2 or A37.3, wherein the one or moremyosin II inhibitors comprise blebbistatin.

A38. The method of any one of embodiments A1 to A37.4, furthercomprising increasing intracellular cyclic adenosine monophosphate(cAMP) levels in the epithelial cells during the culturing in (a).

A39. The method of embodiment A38, wherein increasing intracellularcyclic adenosine monophosphate (cAMP) levels comprises use of one ormore beta-adrenergic agonists and/or one or more beta-adrenergicreceptor agonists.

A39.1 The method of embodiment A39, where the one or morebeta-adrenergic agonists and/or the one or more beta-adrenergic receptoragonists comprise isoproterenol.

A40. The method of any one of embodiments A1 to A39.1, wherein theepithelial cells are obtained from a subject prior to (a).

A40.1 The method of embodiment A40, wherein the subject is a mammal.

A40.2 The method of embodiment A40, wherein the subject is a human.

A40.3 The method of any one of embodiments A40 to A40.2, wherein theepithelial cells are from tissue from a subject.

A40.4 The method of embodiment A40.3, wherein the epithelial cells arefrom differentiated tissue from a subject.

A40.5 The method of any one of embodiments A40 to A40.2, wherein theepithelial cells are from circulating cells from a subject.

A41. The method of any one of embodiments A40 to A40.5, wherein theepithelial cells comprise primary cells from a subject.

A42. The method of any one of embodiments A40 to A40.5, wherein theepithelial cells do not comprise primary cells from a subject.

A43. The method of any one of embodiments A40 to A42, wherein theepithelial cells comprise tumor cells from a subject.

A44. The method of any one of embodiments A41 to A43, wherein theepithelial cells from a subject are selected from squamous cells,columnar cells, adenomatous cells and transitional epithelial cells.

A44.1 The method of any one of embodiments A41 to A43, wherein theepithelial cells from a subject comprise one or more of squamous cells,columnar cells, adenomatous cells and transitional epithelial cells.

A45. The method of any one of embodiments A41 to A44.1, wherein theepithelial cells from a subject comprise keratinocyte epithelial cells.

A45.1 The method of embodiment A45, wherein the keratinocyte epithelialcells are selected from dermal keratinocytes, ocular epithelial cells,corneal epithelial cells, oral mucosal epithelial cells, esophagusepithelial cells, and cervix epithelial cells.

A46. The method of any one of embodiments A41 to A44, wherein theepithelial cells from a subject comprise non-keratinocyte epithelialcells.

A47. The method of embodiment A46, wherein the non-keratinocyteepithelial cells comprise glandular epithelial cells.

A48. The method of embodiment A46 or A47, wherein the non-keratinocyteepithelial cells are selected from prostate cells, mammary cells,hepatocytes, liver epithelial cells, biliary epithelial cells, gallbladder cells, pancreatic islet cells, pancreatic beta cells, pancreaticductal epithelial cells, pulmonary epithelial cells, airway epithelialcells, nasal epithelial cells, kidney cells, bladder cells, urethralepithelial cells, stomach epithelial cells, large intestinal epithelialcells, small intestinal epithelial cells, testicular epithelial cells,ovarian epithelial cells, fallopian tube epithelial cells, thyroidcells, parathyroid cells, adrenal cells, thymus cells, pituitary cells,glandular cells, amniotic epithelial cells, retinal pigmented epithelialcells, sweat gland epithelial cells, sebaceous epithelial cells and hairfollicle cells.

A48.1 The method of any one of embodiments A1 to A47, wherein theepithelial cells comprise basal epithelial cells.

A48.2 The method of any one of embodiments A1 to A47, wherein theepithelial cells are not intestinal epithelial cells.

A49. The method of any one of embodiments A1 to A48.2, wherein theculturing in (a) is performed in the presence of a serum-free medium.

A49.1 The method of embodiment A49, wherein the serum-free medium is adefined serum-free medium.

A49.2 The method of embodiment A49, wherein the serum-free medium is axeno-free serum-free medium.

A49.3 The method of embodiment A49, wherein the serum-free medium is adefined xeno-free serum-free medium.

A50. The method of any one of embodiments A49 to A49.3, wherein theserum-free medium comprises calcium.

A51. The method of embodiment A50, wherein the serum-free mediumcomprises calcium at a concentration below 1 mM.

A52. The method of embodiment A50, wherein the serum-free mediumcomprises calcium at a concentration below 500 μM.

A53. The method of embodiment A50, wherein the serum-free mediumcomprises calcium at a concentration below 100 μM.

A53.1 The method of embodiment A53, wherein the serum-free mediumcomprises calcium at a concentration of about 90 μM.

A54. The method of embodiment A50, wherein the serum-free mediumcomprises calcium at a concentration below 20 μM.

A54.1 The method of any one of embodiments A49 to A54, wherein theserum-free medium comprises a buffer and one or more components selectedfrom inorganic acids, salts, alkali silicates, amino acids, vitamins,purines, pyrimidines, polyamines, alpha-keto acids, organosulphurcompounds and glucose.

A54.2 The method of embodiment A54.1, wherein the one or more salts areselected from sodium chloride, potassium chloride, sodium acetate, andsodium phosphate.

A54.3 The method of embodiment A54.1 or A54.2, wherein the one or moreamino acids are selected from arginine and glutamine.

A54.4 The method of any one of embodiments A54.1 to A54.3, wherein thebuffer is HEPES buffer.

A54.5 The method any one of embodiments A49 to A54.4, wherein theserum-free medium comprises albumin.

A54.6 The method of embodiment A54.5, wherein the albumin is selectedfrom bovine serum albumin and recombinant human serum albumin.

A54.7 The method any one of embodiments A49 to A54.6, wherein theserum-free medium comprises one or more lipids.

A54.8 The method of embodiment A54.7, wherein the one or more lipids areselected from arachidonic acid, cholesterol, DL-alpha-tocopherolacetate, linoleic acid, linolenic acid, myristic acid, oleic acid,palmitic acid, palmitoleic acid, pluronic F-68, stearic acid, andpolysorbate 80.

A54.9 The method of embodiment A54.8, wherein the one or more lipids areselected from linoleic acid, linolenic acid, oleic acid, palmitic acid,and stearic acid.

A55. The method of any one of embodiments A1 to A54.9, which comprisesuse of one or more mitogenic growth factors.

A56. The method of embodiment A55, wherein the one or more mitogenicgrowth factors comprise EGF.

A56.1 The method of embodiment A55, wherein the one or more mitogenicgrowth factors comprise FGF.

A56.2 The method of embodiment A55, wherein the one or more mitogenicgrowth factors comprise EGF and FGF.

A56.3 The method of embodiment A56.1 or A56.2, wherein the FGF comprisesacidic FGF.

A57. The method of any one of embodiments A1 to A54.9, which does notcomprise use of a mitogenic growth factor.

A58. The method of any one of embodiments A1 to A57, which comprises useof one or more mitogenic supplements.

A59. The method of embodiment A58, wherein the one or more mitogenicsupplements comprise bovine pituitary extract (BPE).

A59.1 The method of any one of embodiments A1 to A57, which does notcomprise use of a mitogenic supplement.

A60. The method of any one of embodiments A1 to A59.1, which does notcomprise use of a Wnt agonist or a beta-catenin agonist.

A60.1 The method of any one of embodiments A1 to A60, which does notcomprise use of one or more of components selected from: noggin,R-spondin, Wnt-3a, EGF, nicotinamide, FGF10, gastrin, a p38 inhibitor,SB202190, DHT, a notch inhibitor, a gamma secretase inhibitor, DBZ andDAPT.

A61. The method of any one of embodiments A1 to A60.1, which does notcomprise use of an extracellular matrix.

A62. The method of any one of embodiments A1 to A60.1, wherein theculturing in (a) is performed in a container comprising a coating.

A63. The method of embodiment A62, wherein the coating comprisescollagen.

A64. The method of embodiment A62, wherein the coating comprises abasement membrane matrix.

A65. The method of any one of embodiments A1 to A64, wherein theculturing in (a) comprises expanding the epithelial cells.

A66. The method of embodiment A65, wherein the epithelial cells areexpanded at least about 2-fold.

A67. The method of embodiment A65, wherein the epithelial cells areexpanded at least about 5-fold.

A68. The method of embodiment A65, wherein the epithelial cells areexpanded at least about 10-fold.

A69. The method of embodiment A65, wherein the epithelial cells areexpanded at least about 15-fold.

A70. The method of embodiment A65, wherein the epithelial cells areexpanded at least about 20-fold.

A70.1 The method of embodiment A65, wherein the epithelial cells areexpanded at least about 100-fold.

A70.2 The method of embodiment A65, wherein the epithelial cells areexpanded at least about 1,000-fold.

A70.3 The method of embodiment A65, wherein the epithelial cells areexpanded at least about 10,000-fold.

A70.4 The method of embodiment A65, wherein the epithelial cells areexpanded at least about 100,000-fold.

A70.5 The method of embodiment A65, wherein the epithelial cells areexpanded at least about 1 million-fold.

A70.6 The method of embodiment A65, wherein the epithelial cells areexpanded at least about 1 billion-fold.

A70.7 The method of embodiment A65, wherein the epithelial cells areexpanded at least about 1 trillion-fold.

A71. The method of any one of embodiments A65 to A70.7, wherein theepithelial cells are cultured for about 4 days.

A72. The method of any one of embodiments A65 to A70.7, wherein theepithelial cells are cultured for about 5 days.

A73. The method of any one of embodiments A1 to A72, wherein theepithelial cells are continuously proliferated.

A74. The method of any one of embodiments A1 to A73, comprisingpassaging the epithelial cells at least 15 times.

A75. The method of any one of embodiments A1 to A73, comprisingpassaging the epithelial cells at least 25 times.

A76. The method of any one of embodiments A1 to A75, wherein apopulation of the epithelial cells doubles over a period of time.

A77. The method of embodiment A76, wherein the epithelial cellpopulation doubles at least 20 times.

A78. The method of embodiment A76, wherein the epithelial cellpopulation doubles at least 50 times.

A78.1 The method of embodiment A76, wherein the epithelial cellpopulation doubles at least 80 times.

A79. The method of embodiment A76, wherein the epithelial cellpopulation doubles at least 100 times.

A80. The method of embodiment A76, wherein the epithelial cellpopulation doubles at least 120 times.

A81. The method of embodiment A76, wherein the epithelial cellpopulation doubles at least 150 times.

A82. The method of embodiment A76, wherein the epithelial cellpopulation doubles at least 200 times.

A83. The method of any one of embodiments A76 to A82, wherein the periodof time is about 50 days.

A84. The method of any one of embodiments A76 to A82, wherein the periodof time is about 100 days.

A85. The method of any one of embodiments A76 to A82, wherein the periodof time is about 150 days.

A86. The method of any one of embodiments A76 to A82, wherein the periodof time is about 200 days.

A87. The method of any one of embodiments A1 to A86, wherein theepithelial cells maintain one or more native functional characteristicsduring (b).

A88. The method of any one of embodiments A1 to A86, wherein theepithelial cells do not maintain one or more native functionalcharacteristics during (b).

A89. The method of any one of embodiments A1 to A88, wherein theepithelial cells are placed after (b) into a cell culture environmentwherein TGF-beta signaling is not inhibited.

A90. The method of embodiment A89, wherein the epithelial cells maintainor regain one or more native functional characteristics after placementinto the cell culture environment wherein TGF-beta signaling is notinhibited.

A91. The method of any one of embodiments A1 to A90, wherein theepithelial cells can be induced to differentiate into multiple tissuetypes.

A91.1 The method of any one of embodiments A1 to A90, wherein theepithelial cells do not acquire the ability to differentiate intomultiple tissue types.

A92. The method of any one of embodiments A1 to A91.1, wherein theepithelial cells do not acquire the ability to form organoids.

A93. The method of any one of embodiments A1 to A92, wherein theepithelial cells are not derived from embryonic stem cells.

A93.1 The method of any one of embodiments A1 to A93, wherein theepithelial cells are not derived from continuously proliferatingepithelial stem cells.

A93.2 The method of any one of embodiments A1 to A93.1, wherein theepithelial cells are derived from epithelial tissue comprising quiescentepithelial cells.

A93.3 The method of any one of embodiments A1 to A93.2, which methoddoes not comprise selecting for continuously proliferating epithelialstem cells.

A93.4 The method of any one of embodiments A1 to A93.3, which methoddoes not comprise selecting for intestinal crypt cells.

A93.5 The method of any one of embodiments A1 to A93.4, which methoddoes not comprise selecting for LGR5+ cells.

A94. The method of any one of embodiments A40 to A93.5, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture do not comprise continuously proliferating epithelial stem cellsor cells derived from continuously proliferating epithelial stem cells.

A94.1 The method of any one of embodiments A40 to A94, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture do not comprise pluripotent stem cells or cells derived frompluripotent stem cells.

A94.2 The method of any one of embodiments A40 to A94.1, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture do not comprise terminally differentiated epithelial cells.

A94.3 The method of any one of embodiments A40 to A94.2, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture do not comprise gastric epithelial cells, intestinal epithelialcells, and/or pancreatic epithelial cells.

A94.4 The method of any one of embodiments A40 to A94.3, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture do not comprise intestinal crypt cells.

A94.5 The method of any one of embodiments A40 to A94.4, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture do not comprise LGR5+ cells.

A95. The method of any one of embodiments A40 to A94.5, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture are a homogenous population of epithelial cells.

A95.1 The method of any one of embodiments A40 to A95, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture are a homogenous population of basal epithelial cells.

A95.2 The method of any one of embodiments A40 to A94.5, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture are a heterogeneous population of epithelial cells.

A96. The method of any one of embodiments A40 to A95.2, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture are less differentiated than terminally differentiated cells andare more differentiated than embryonic stem cells or adult stem cells.

A97. The method of any one of embodiments A40 to A96, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture express one or more basal epithelial cell markers.

A98. The method of any one of embodiments A40 to A97, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture express one or more of ITGA6, ITGB4, KRT14, KRT15, KRT5 andTP63.

A99. The method of any one of embodiments A40 to A98, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture do not express one or more epithelial stem cell markers.

A100. The method of any one of embodiments A40 to A99, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture do not express Lgr5.

A101. The method of any one of embodiments A40 to A100, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture do not express one or more pluripotent stem cell markers.

A102. The method of any one of embodiments A40 to A101, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture do not express one or more of LIN28A, NANOG, POU5F1/OCT4 andSOX2.

A103. The method of any one of embodiments A40 to A102, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture do not express one or more terminally differentiated epithelialcell markers.

A104. The method of any one of embodiments A40 to A103, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture do not express one or more of CFTR, FOXJ1, IVL, KRT1, KRT10,KRT20, LOR, MUC1, MUC5AC, SCGB1A1, SFTPB and SFTPD.

A105. The method of any one of embodiments A40 to A104, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture do not express one or more gastric epithelial cell markers, oneor more intestinal epithelial cell markers, and/or one or morepancreatic epithelial cell markers.

A106. The method of any one of embodiments A40 to A105, wherein theepithelial cells obtained from a subject and/or the epithelial cells inculture do not express one or more of CD34, HNF1 A, HNF4A, IHH, KIT,LGR5, PDX1, and PROM1/CD133.

A107. The method of any one of embodiments A1 to A106, furthercomprising isolating a population of ex vivo proliferated epithelialcells.

A108. The method of any one of embodiments A1 to A107, furthercomprising storing a population of ex vivo proliferated epithelial cellsin a cell bank.

A109. A population of ex vivo proliferated epithelial cells produced bya method according to any one of embodiments A1 to A108.

A110. Use of the population of ex vivo proliferated epithelial cells ofembodiment A109 for production of genetically modified cells.

A111. Use of the population of ex vivo proliferated epithelial cells ofembodiment A109 for identifying one or more candidate treatments for asubject.

A112. Use of the population of ex vivo proliferated epithelial cells ofembodiment A109 for identifying one or more abnormal epithelial cells ina subject.

A113. Use of the population of ex vivo proliferated epithelial cells ofembodiment A109 for monitoring the progression of a disease or treatmentof a disease in a subject.

B1. A serum-free cell culture medium for proliferating differentiatedepithelial cells ex vivo under feeder-cell free conditions, whichserum-free medium comprises one or more TGF-beta inhibitors and/or oneor more TGF-beta signaling inhibitors.

B1.1 A serum-free cell culture medium for proliferating formerlyquiescent epithelial cells ex vivo under feeder-cell free conditions,which serum-free medium comprises one or more TGF-beta inhibitors and/orone or more TGF-beta signaling inhibitors.

B1.2 A serum-free cell culture medium for proliferatinglineage-committed epithelial cells ex vivo under feeder-cell freeconditions, which serum-free medium comprises one or more TGF-betainhibitors and/or one or more TGF-beta signaling inhibitors.

B1.3 A serum-free cell culture medium for proliferating differentiatedepithelial cells ex vivo under feeder-cell free conditions, whichserum-free medium comprises a small molecule inhibitor consisting of aTGF-beta inhibitor or a TGF-beta signaling inhibitor.

B1.4 A serum-free cell culture medium for proliferating formerlyquiescent epithelial cells ex vivo under feeder-cell free conditions,which serum-free medium comprises a small molecule inhibitor consistingof a TGF-beta inhibitor or a TGF-beta signaling inhibitor.

B1.5 A serum-free cell culture medium for proliferatinglineage-committed epithelial cells ex vivo under feeder-cell freeconditions, which serum-free medium comprises a small molecule inhibitorconsisting of a TGF-beta inhibitor or a TGF-beta signaling inhibitor.

B1.6 The serum-free cell culture medium of any one of embodiments B1 toB1.5, which is a defined serum-free cell culture medium.

B1.7 The serum-free cell culture medium of any one of embodiments B1 toB1.5, which is a xeno-free serum-free cell culture medium.

B1.8 The serum-free cell culture medium of any one of embodiments B1 toB1.5, which is a defined xeno-free serum-free cell culture medium.

B2. A cell culture medium for proliferating epithelial cells ex vivounder feeder-cell free conditions, which medium comprises one or moreTGF-beta inhibitors and/or one or more TGF-beta signaling inhibitors,and one or more PAK1 inhibitors.

B2.1 A cell culture medium for proliferating epithelial cells ex vivounder feeder-cell free conditions, which medium comprises small moleculeinhibitors consisting of a TGF-beta inhibitor or a TGF-beta signalinginhibitor and a PAK1 inhibitor.

B3. A cell culture medium for proliferating epithelial cells ex vivounder feeder-cell free conditions, which medium comprises one or moreTGF-beta inhibitors and/or one or more TGF-beta signaling inhibitors,and one or more myosin II inhibitors.

B3.1 The cell culture medium of embodiment B3, wherein the myosin II isa non-muscle myosin II (NM II).

B3.2 A cell culture medium for proliferating epithelial cells ex vivounder feeder-cell free conditions, which medium comprises small moleculeinhibitors consisting of a TGF-beta inhibitor or a TGF-beta signalinginhibitor and a myosin II inhibitor.

B3.3 The cell culture medium of embodiment B3.2, wherein the myosin IIis a non-muscle myosin II (NM II).

B4. The cell culture medium of any one of embodiments B2 to B3.3,wherein the epithelial cells comprise differentiated epithelial cells.

B4.1 The cell culture medium of any one of embodiments B2 to B3.3,wherein the epithelial cells comprise formerly quiescent epithelialcells.

B4.2 The cell culture medium of any one of embodiments B2 to B3.3,wherein the epithelial cells comprise lineage-committed epithelialcells.

B5. The cell culture medium of any one of embodiments B2 to B4, which isa serum containing medium.

B5.1 The cell culture medium of any one of embodiments B2 to B4, whichis a serum-free medium.

B5.2 The cell culture medium of embodiment B5.1, which is a definedserum-free medium.

B5.3 The cell culture medium of embodiment B5.1, which is a xeno-freeserum-free medium.

B5.4 The cell culture medium of embodiment B5.1, which is a definedxeno-free serum-free medium.

B6. The cell culture medium of any one of embodiments B1 to B5.3,wherein the one or more TGF-beta inhibitors and/or the one or moreTGF-beta signaling inhibitors bind to one or more TGF-beta receptors orone or more TGF-beta ligands or both.

B7. The cell culture medium of any one of embodiments B1 to B6, whereinthe one or more TGF-beta inhibitors and/or the one or more TGF-betasignaling inhibitors disrupt one or more TGF-beta receptor-ligandinteractions.

B8. The cell culture medium of any one of embodiments B1 to B7, whereinthe one or more TGF-beta inhibitors and/or the one or more TGF-betasignaling inhibitors do not comprise a recombinant protein.

B9. The cell culture medium of any one of embodiments B1 to B8, whereinthe one or more TGF-beta inhibitors and/or the one or more TGF-betasignaling inhibitors do not comprise Noggin, DAN, Cerberus or Gremlin.

B10. The cell culture medium of any one of embodiments B1 to B9, whereinthe one or more TGF-beta inhibitors and/or the one or more TGF-betasignaling inhibitors comprise one or more TGF-beta receptor inhibitors.

B11. The cell culture medium of embodiment B10, wherein the one or moreTGF-beta receptor inhibitors comprise one or more TGF-beta type Ireceptor inhibitors.

B12. The cell culture medium of embodiment B11, wherein the one or moreTGF-beta type I receptor inhibitors are selected from an ALK1 inhibitor,an ALK2 inhibitor, an ALK3 inhibitor, an ALK4 inhibitor, an ALK5inhibitor, an ALK6 inhibitor, an ALK7 inhibitor, and an ALK8 inhibitor.

B13. The cell culture medium of embodiment B12, wherein the one or moreTGF-beta type I receptor inhibitors comprise one or more ALK5inhibitors.

B14. The cell culture medium of embodiment B13, wherein the one or moreALK5 inhibitors comprise one or more small molecule ALK5 inhibitors.

B15. The cell culture medium of embodiment B15, wherein the one or moreALK5 inhibitors comprise one or more ATP analogs.

B16. The cell culture medium of embodiment B14 or B15, wherein at leastone of the one or more ALK5 inhibitors comprises the structure ofFormula A:

wherein:

-   -   X, Y and Z independently are chosen from N, C and O;    -   R¹, R² and R³ independently are chosen from hydrogen, C1-C10        alkyl, substituted C1-C10 alkyl, C3-C9 cycloalkyl, substituted        C3-C9 cycloalkyl, C5-C10 aryl, substituted C5-C10 aryl, C5-C10        cycloaryl, substituted C5-C10 cycloaryl, C5-C9 heterocyclic,        substituted C5-C9 heterocyclic, C5-C9 hetercycloaryl,        substituted C5-C9 heterocycloaryl, -linker-(C3-C9 cycloalkyl),        -linker-(substituted C3-C9 cycloalkyl), -linker-(C5-C10 aryl),        -linker-(substituted C5-C10 aryl), -linker-(C5-C10 cycloaryl),        -linker-(substituted C5-C10 cycloaryl), -linker-(C5-C9        heterocyclic), -linker-(substituted C5-C9 heterocyclic),        -linker-(C5-C9 hetercycloaryl), -linker-(substituted C5-C9        heterocycloaryl);    -   n is 0 or 1;    -   R⁴, R⁵ and R⁶ independently are chosen from hydrogen, C1-C10        alkyl, substituted C1-C10 alkyl, C1-C10 alkoxy, substituted        C1-C10 alkoxy, C1-C6 alkanoyl, C1-C6 alkoxycarbonyl, substituted        C1-C6 alkanoyl, substituted C1-C6 alkoxycarbonyl, C3-C9        cycloalkyl, substituted C3-C9 cycloalkyl, C5-C10 aryl,        substituted C5-C10 aryl, C5-C10 cycloaryl, substituted C5-C10        cycloaryl, C5-C9 heterocyclic, substituted C5-C9 heterocyclic,        C5-C9 hetercycloaryl, substituted C5-C9 heterocycloaryl,        -linker-(C3-C9 cycloalkyl), -linker-(substituted C3-C9        cycloalkyl), -linker-(C5-C10 aryl), -linker-(substituted C5-C10        aryl), -linker-(C5-C10 cycloaryl), -linker-(substituted C5-C10        cycloaryl), -linker-(C5-C9 heterocyclic), -linker-(substituted        C5-C9 heterocyclic), -linker-(C5-C9 hetercycloaryl),        -linker-(substituted C5-C9 heterocycloaryl); and    -   the substituents on the substituted alkyl, alkoxy, alkanoyl,        alkoxycarbonyl cycloalkyl, aryl, cycloaryl, heterocyclic or        heterocycloaryl groups are hydroxyl, C1-C10 alkyl, hydroxyl        C1-C10 alkylene, C1-C6 alkoxy, C3-C9 cycloalkyl, C5-C9        heterocyclic, C1-6 alkoxy C1-6 alkenyl, amino, cyano, halogen or        aryl.

B17. The cell culture medium of embodiment B14, B15 or B16, wherein theone or more ALK5 inhibitors are selected from A83-01, GW788388, RepSox,and SB 431542.

B18. The cell culture medium of embodiment B17, wherein the one or moreALK5 inhibitors comprise A83-01.

B19. The cell culture medium of any one of embodiments B13 to B18,wherein the one or more ALK5 inhibitors binds to ALK5 or one or moreALK5 ligands or both.

B20. The cell culture medium of any one of embodiments B13 to B19,wherein the one or more ALK5 inhibitors disrupt one or more ALK5-ligandinteractions.

B21. The cell culture medium of any one of embodiments B1 to B20,further comprising one or more Rho kinase inhibitors and/or one or moreRho-associated protein kinase inhibitors.

B22. The cell culture medium of embodiment B21, wherein the one or moreRho kinase inhibitors and/or the one or more Rho-associated proteinkinase inhibitors are selected from a Rho kinase 1 (ROCK 1) inhibitorand a Rho kinase 2 (ROCK 2) inhibitor.

B23. The cell culture medium of embodiment B21 or B22, wherein the oneor more Rho kinase inhibitors and/or the one or more Rho-associatedprotein kinase inhibitors comprise one or more small molecule Rho kinaseinhibitors and/or one or more small molecule Rho-associated proteinkinase inhibitors.

B24. The cell culture medium of embodiment B23, wherein the one or moreRho kinase inhibitors and/or the one or more Rho-associated proteinkinase inhibitors is selected from Y-27632, SR 3677, thiazovivin, HA1100hydrochloride, HA1077 and GSK-429286.

B25. The cell culture medium of embodiment B14, wherein the one or moreRho kinase inhibitors and/or the one or more Rho-associated proteinkinase inhibitors comprise Y-27632.

B25.1 The cell culture medium of any one of embodiments B1 to B20, whichdoes not comprise a Rho kinase inhibitor and/or a Rho-associated proteinkinase inhibitor.

B26. The cell culture medium of any one of embodiments B1 to B25.1,further comprising one or more PAK1 inhibitors.

B27. The cell culture medium of embodiment B26, wherein the one or morePAK1 inhibitors comprise one or more small molecule PAK1 inhibitors.

B28. The cell culture medium of embodiment B27, wherein the one or morePAK1 inhibitors comprise IPA3.

B29. The cell culture medium of any one of embodiments B1 to B28,further comprising one or more myosin II inhibitors.

B29.1 The cell culture medium of embodiment B29, wherein the one or moremyosin II inhibitors comprise one or more non-muscle myosin II (NM II)inhibitors.

B30. The cell culture medium of embodiment B29 or B29.1, wherein the oneor more myosin II inhibitors comprise one or more small molecule myosinII inhibitors.

B30.1 The cell culture medium of embodiment B30, wherein the one or moremyosin II inhibitors comprise blebbistatin.

B31. The cell culture medium of any one of embodiments B1 to B30.1,further comprising one or more beta-adrenergic agonists and/or one ormore beta-adrenergic receptor agonists.

B31.1 The cell culture medium of embodiment B31, wherein the one or morebeta-adrenergic agonists and/or the one or more beta-adrenergic receptoragonists comprise isoproterenol.

B32. The cell culture medium of any one of embodiments B1 to B31.1,wherein the epithelial cells are from a subject.

B32.1 The cell culture medium of embodiment B32, wherein the epithelialcells are from tissue from a subject.

B32.2 The cell culture medium of embodiment B32.1, wherein theepithelial cells are from differentiated tissue from a subject.

B32.3 The cell culture medium of any one of embodiments B32 to B32.2,wherein the epithelial cells comprise primary cells.

B33. The cell culture medium of any one of embodiments B32 to B32.2,wherein the epithelial cells do not comprise primary cells.

B34. The cell culture medium of any one of embodiments B32 to B33,wherein the epithelial cells comprise tumor cells.

B35. The cell culture medium of any one of embodiments B32 to B34,wherein the epithelial cells from a subject are selected from squamouscells, columnar cells, adenomatous cells and transitional epithelialcells.

B35.1 The cell culture medium of any one of embodiments B32 to B34,wherein the epithelial cells from a subject comprise one or more ofsquamous cells, columnar cells, adenomatous cells and transitionalepithelial cells.

B36. The cell culture medium of any one of embodiments B32 to B35.1,wherein the epithelial cells from a subject comprise keratinocyteepithelial cells.

B36.1 The cell culture medium of embodiment B36, wherein thekeratinocyte epithelial cells are selected from dermal keratinocyte,ocular epithelial cells, corneal epithelial cells, oral mucosalepithelial cells, esophagus epithelial cells, and cervix epithelialcells.

B37. The cell culture medium of any one of embodiments B32 to B35,wherein the epithelial cells from a subject comprise non-keratinocyteepithelial cells.

B38. The cell culture medium of embodiment B37, wherein thenon-keratinocyte epithelial cells comprise glandular epithelial cells.

B39. The cell culture medium of embodiment B37 or B38, wherein thenon-keratinocyte epithelial cells are selected from prostate cells,mammary cells, hepatocytes, liver epithelial cells, biliary epithelialcells, gall bladder cells, pancreatic islet cells, pancreatic betacells, pancreatic ductal epithelial cells, pulmonary epithelial cells,airway epithelial cells, nasal epithelial cells, kidney cells, bladdercells, urethral epithelial cells, stomach epithelial cells, largeintestinal epithelial cells, small intestinal epithelial cells,testicular epithelial cells, ovarian epithelial cells, fallopian tubeepithelial cells, thyroid cells, parathyroid cells, adrenal cells,thymus cells, pituitary cells, glandular cells, amniotic epithelialcells, retinal pigmented epithelial cells, sweat gland epithelial cells,sebaceous epithelial cells and hair follicle cells.

B39.1 The cell culture medium of any one of embodiments B1 to B39,wherein the epithelial cells comprise basal epithelial cells.

B39.2 The cell culture medium of any one of embodiments B1 to B39,wherein the epithelial cells are not intestinal epithelial cells.

B40. The cell culture medium of any one of embodiments B1 to B39.2,which comprises calcium.

B41. The cell culture medium of embodiment B40, wherein the calcium ispresent at a concentration below 1 mM.

B42. The cell culture medium of embodiment B40, wherein the calcium ispresent at a concentration below 500 μM.

B43. The cell culture medium of embodiment B40, wherein the calcium ispresent at a concentration below 100 μM.

B43.1 The cell culture medium of embodiment B43, wherein the calcium ispresent at a concentration of about 90 μM.

B44. The cell culture medium of embodiment B40, wherein the calcium ispresent at a concentration below 20 μM.

B44.1 The cell culture medium of any one of embodiments B1 to B44, whichcomprises a buffer and one or more components selected from inorganicacids, salts, alkali silicates, amino acids, vitamins, purines,pyrimidines, polyamines, alpha-keto acids, organosulphur compounds andglucose.

B44.2 The cell culture medium of embodiment B44.1, wherein the one ormore salts are selected from sodium chloride, potassium chloride, sodiumacetate, and sodium phosphate.

B44.3 The cell culture medium of embodiment B44.1 or B44.2, wherein theone or more amino acids are selected from arginine and glutamine.

B44.4 The cell culture medium of any one of embodiments B44.1 to B44.3,wherein the buffer is HEPES buffer.

B44.5 The cell culture medium any one of embodiments B1 to B44.4, whichcomprises albumin.

B44.6 The cell culture medium of embodiment B44.5, wherein the albuminis selected from bovine serum albumin and recombinant human serumalbumin.

B44.7 The cell culture medium any one of embodiments B1 to B44.6, whichcomprises one or more lipids.

B44.8 The cell culture medium of embodiment B44.7, wherein the one ormore lipids are selected from arachidonic acid, cholesterol,DL-alpha-tocopherol acetate, linoleic acid, linolenic acid, myristicacid, oleic acid, palmitic acid, palmitoleic acid, pluronic F-68,stearic acid, and polysorbate 80.

B44.9 The cell culture medium of embodiment B44.8, wherein the one ormore lipids are selected from linoleic acid, linolenic acid, oleic acid,palmitic acid, and stearic acid.

B45. The cell culture medium of any one of embodiments B1 to B44.9,which comprises one or more mitogenic growth factors.

B46. The cell culture medium of embodiment B45, wherein the one or moremitogenic growth factors comprise EGF.

B46.1 The cell culture medium of embodiment B45, wherein the one or moremitogenic growth factors comprise FGF.

B46.2 The cell culture medium of embodiment B45, wherein the one or moremitogenic growth factors comprise EGF and FGF.

B46.3 The cell culture medium of embodiment B46.1 or B46.2, wherein theFGF comprises acidic FGF.

B47. The cell culture medium of any one of embodiments B1 to B44.9,which does not comprise a mitogenic growth factor.

B48. The cell culture medium of any one of embodiments B1 to B47, whichcomprises one or more mitogenic supplements.

B49. The cell culture medium of embodiment B48, wherein the one or moremitogenic supplements comprise bovine pituitary extract (BPE).

B49.1 The cell culture medium of any one of embodiments B1 to B47, whichdoes not comprise a mitogenic supplement.

B50. The cell culture medium of any one of embodiments B1 to B49.1,which does not comprise a Wnt agonist or a beta-catenin agonist.

B50.1 The cell culture medium of any one of embodiments B1 to B50, whichdoes not comprise one or more of components selected from: noggin,R-spondin, Wnt-3a, EGF, nicotinamide, FGF10, gastrin, a p38 inhibitor,SB202190, DHT, a notch inhibitor, a gamma secretase inhibitor, DBZ andDAPT.

B51. The cell culture medium of any one of embodiments B1 to B50.1,which does not comprise an extracellular matrix.

C1. A population of ex vivo proliferated epithelial cells produced by amethod comprising:

-   -   a) culturing differentiated epithelial cells under serum-free        and feeder-cell free conditions; and    -   b) inhibiting TGF-beta signaling in the differentiated        epithelial cells during the culturing in (a).

C1.1 A population of ex vivo proliferated epithelial cells produced by amethod comprising:

-   -   a) culturing formerly quiescent epithelial cells under        serum-free and feeder-cell free conditions; and    -   b) inhibiting TGF-beta signaling in the formerly quiescent        epithelial cells during the culturing in (a).

C1.2 A population of ex vivo proliferated epithelial cells produced by amethod comprising:

-   -   a) culturing lineage-committed epithelial cells under serum-free        and feeder-cell free conditions; and    -   b) inhibiting TGF-beta signaling in the lineage-committed        epithelial cells during the culturing in (a).

C2. A population of ex vivo proliferated epithelial cells produced by amethod comprising:

-   -   a) culturing epithelial cells under feeder-cell free conditions;    -   b) inhibiting TGF-beta signaling in the epithelial cells during        the culturing in (a); and    -   c) inhibiting the activity of p21-activated kinase (PAK) in the        epithelial cells during the culturing in (a).

C2.1 The epithelial cells of embodiment C2, which comprisedifferentiated epithelial cells.

C2.2 The epithelial cells of embodiment C2, which comprise formerlyquiescent epithelial cells.

C2.3 The epithelial cells of embodiment C2, which compriselineage-committed epithelial cells.

C3. A population of ex vivo proliferated epithelial cells produced by amethod comprising:

-   -   a) culturing epithelial cells under serum-free and feeder-cell        free conditions;    -   b) inhibiting TGF-beta signaling in the epithelial cells during        the culturing in (a); and    -   c) inhibiting the activity of myosin II in the epithelial cells        during the culturing in (a).

C3.1 The epithelial cells of embodiment C3, which comprisedifferentiated epithelial cells.

C3.2 The epithelial cells of embodiment C3, which comprise formerlyquiescent epithelial cells.

C3.3 The epithelial cells of embodiment C3, which compriselineage-committed epithelial cells.

C3.4 The epithelial cells of any one or embodiments C3 to C3.3, whereinthe myosin II is a non-muscle myosin II (NM II).

C4. The epithelial cells of any one of embodiments C2 to C3.4, whereinthe culturing in (a) is performed in the presence of a serum containingmedium.

C5. The epithelial cells of any one of embodiments C2 to C3.4, whereinthe culturing in (a) is performed in the presence of a serum-freemedium.

C5.1 The epithelial cells of any one of embodiments C1 to C5, wherein(a) and (b) are performed at the same time; or (a), (b) and (c) areperformed at the same time.

C5.2 The epithelial cells of any one of embodiments C1 to C5.1, whereinthe epithelial cells are frozen and thawed prior to (a).

C6. The epithelial cells of any one of embodiments C1 to C5.2, whereinthe activity of one or more TGF-beta receptors is inhibited in (b).

C7. The epithelial cells of embodiment C6, wherein one or more TGF-betareceptor-ligand interactions are inhibited in (b).

C8. The epithelial cells of embodiment C6 or C7, wherein the one or moreTGF-beta receptors comprise a TGF-beta type I receptor.

C9. The epithelial cells of embodiment C8, wherein the TGF-beta type Ireceptor is selected from ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, ALK7 andALK8.

C10. The epithelial cells of embodiment C8, wherein the one or moreTGF-beta receptors comprise ALK5.

C11. The epithelial cells of any one of embodiments C1 to C10, whereininhibiting TGF-beta signaling comprises use of one or more TGF-betainhibitors and/or one or more TGF-beta signaling inhibitors.

C12. The epithelial cells of embodiment C11, wherein the one or moreTGF-beta inhibitors and/or the one or more TGF-beta signaling inhibitorsbind to one or more TGF-beta receptors or one or more TGF-beta ligandsor both.

C13. The epithelial cells embodiment C11 or C12, wherein the one or moreTGF-beta inhibitors and/or the one or more TGF-beta signaling inhibitorsdisrupt one or more TGF-beta receptor-ligand interactions.

C14. The epithelial cells of embodiment C11, C12 or C13, wherein the oneor more TGF-beta inhibitors and/or the one or more TGF-beta signalinginhibitors do not comprise a recombinant protein.

C15. The epithelial cells of any one of embodiments C11 to C14, whereinthe one or more TGF-beta inhibitors and/or the one or more TGF-betasignaling inhibitors do not comprise Noggin, DAN, Cerberus or Gremlin.

C16. The epithelial cells of any one of embodiments C11 to C15, whereinthe one or more TGF-beta inhibitors and/or the one or more TGF-betasignaling inhibitors comprise one or more ALK5 inhibitors.

C17. The epithelial cells of embodiment C16, wherein the one or moreALK5 inhibitors comprise one or more small molecule ALK5 inhibitors.

C18. The epithelial cells of embodiment C17, wherein the one or moreALK5 inhibitors comprise one or more ATP analogs.

C19. The epithelial cells of any one of embodiments C16 to C18, whereinat least one of the one or more ALK5 inhibitors comprises the structureof Formula A:

wherein:

-   -   X, Y and Z independently are chosen from N, C and O;    -   R¹, R² and R³ independently are chosen from hydrogen, C1-C10        alkyl, substituted C1-C10 alkyl, C3-C9 cycloalkyl, substituted        C3-C9 cycloalkyl, C5-C10 aryl, substituted C5-C10 aryl, C5-C10        cycloaryl, substituted C5-C10 cycloaryl, C5-C9 heterocyclic,        substituted C5-C9 heterocyclic, C5-C9 hetercycloaryl,        substituted C5-C9 heterocycloaryl, -linker-(C3-C9 cycloalkyl),        -linker-(substituted C3-C9 cycloalkyl), -linker-(C5-C10 aryl),        -linker-(substituted C5-C10 aryl), -linker-(C5-C10 cycloaryl),        -linker-(substituted C5-C10 cycloaryl), -linker-(C5-C9        heterocyclic), -linker-(substituted C5-C9 heterocyclic),        -linker-(C5-C9 hetercycloaryl), -linker-(substituted C5-C9        heterocycloaryl);    -   n is 0 or 1;    -   R⁴, R⁵ and R⁶ independently are chosen from hydrogen, C1-C10        alkyl, substituted C1-C10 alkyl, C1-C10 alkoxy, substituted        C1-C10 alkoxy, C1-C6 alkanoyl, C1-C6 alkoxycarbonyl, substituted        C1-C6 alkanoyl, substituted C1-C6 alkoxycarbonyl, C3-C9        cycloalkyl, substituted C3-C9 cycloalkyl, C5-C10 aryl,        substituted C5-C10 aryl, C5-C10 cycloaryl, substituted C5-C10        cycloaryl, C5-C9 heterocyclic, substituted C5-C9 heterocyclic,        C5-C9 hetercycloaryl, substituted C5-C9 heterocycloaryl,        -linker-(C3-C9 cycloalkyl), -linker-(substituted C3-C9        cycloalkyl), -linker-(C5-C10 aryl), -linker-(substituted C5-C10        aryl), -linker-(C5-C10 cycloaryl), -linker-(substituted C5-C10        cycloaryl), -linker-(C5-C9 heterocyclic), -linker-(substituted        C5-C9 heterocyclic), -linker-(C5-C9 hetercycloaryl),        -linker-(substituted C5-C9 heterocycloaryl); and    -   the substituents on the substituted alkyl, alkoxy, alkanoyl,        alkoxycarbonyl cycloalkyl, aryl, cycloaryl, heterocyclic or        heterocycloaryl groups are hydroxyl, C1-C10 alkyl, hydroxyl        C1-C10 alkylene, C1-C6 alkoxy, C3-C9 cycloalkyl, C5-C9        heterocyclic, C1-6 alkoxy C1-6 alkenyl, amino, cyano, halogen or        aryl.

C20. The epithelial cells of any one of embodiments C16 to C19, whereinthe one or more ALK5 inhibitors are selected from A83-01, GW788388,RepSox, and SB 431542.

C21. The epithelial cells of embodiment C20, wherein the one or moreALK5 inhibitors comprise A83-01.

C22. The epithelial cells of any one of embodiments C16 to C21, whereinthe one or more ALK5 inhibitors bind to ALK5 or one or more ALK5 ligandsor both.

C23. The epithelial cells of any one of embodiments C16 to C22, whereinthe one or more ALK5 inhibitors disrupt one or more ALK5-ligandinteractions.

C24. The epithelial cells of any one of embodiments C16 to C23, whereinthe method comprises activating telomerase and/or modulatingcytoskeletal structure in the epithelial cells.

C25. The epithelial cells of any one of embodiments C1 to C24, whereinthe method further comprises inhibiting the activity of Rho kinaseand/or Rho-associated protein kinase in the epithelial cells during theculturing in (a).

C26. The epithelial cells of embodiment C25, wherein Rho kinase and/orthe Rho-associated protein kinase is selected from Rho kinase 1 (ROCK 1)and Rho kinase 2 (ROCK 2).

C27. The epithelial cells of embodiment C25 or C26, wherein inhibitingthe activity of Rho kinase and/or Rho-associated protein kinasecomprises use of one or more Rho kinase inhibitors and/or one or moreRho-associated protein kinase inhibitors.

C28. The epithelial cells of embodiment C27, wherein the one or more Rhokinase inhibitors and/or the one or more Rho-associated protein kinaseinhibitors comprise one or more small molecule Rho kinase inhibitors.

C29. The epithelial cells of embodiment C28, wherein the one or more Rhokinase inhibitors and/or the one or more Rho-associated protein kinaseinhibitors is selected from Y-27632, SR 3677, thiazovivin, HA1100hydrochloride, HA1077 and GSK-429286.

C30. The epithelial cells of embodiment C29, wherein the one or more Rhokinase inhibitors and/or the one or more Rho-associated protein kinaseinhibitors comprise Y-27632.

C30.1 The epithelial cells of any one of embodiments C1 to C24, whereinthe method does not comprise inhibiting the activity of Rho kinaseand/or Rho-associated protein kinase in the epithelial cells during theculturing in (a).

C31. The epithelial cells of any one of embodiments C1 to C30.1, whereinthe method further comprises inhibiting the activity of p21-activatedkinase (PAK) in the epithelial cells during the culturing in (a).

C32. The epithelial cells of embodiment C31, wherein the PAK is selectedfrom PAK1, PAK2, PAK3 and PAK4.

C33. The epithelial cells of embodiment C32, wherein the PAK is PAK1.

C34. The epithelial cells of embodiment C33, wherein inhibiting theactivity of PAK1 comprises use of one or more PAK1 inhibitors.

C35. The epithelial cells of embodiment C34, wherein the one or morePAK1 inhibitors comprise one or more small molecule PAK1 inhibitors.

C36. The epithelial cells of embodiment C35, wherein the one or morePAK1 inhibitors comprise IPA3.

C37. The epithelial cells of any one of embodiments C1 to C36, whereinthe method further comprises inhibiting the activity of myosin II in theepithelial cells during the culturing in (a).

C37.1 The epithelial cells of embodiment C37, wherein the myosin II is anon-muscle myosin II (NM II).

C37.2 The epithelial cells of embodiment C37 or C37.1, whereininhibiting the activity of myosin II comprises use of one or more myosinII inhibitors.

C37.3 The epithelial cells of embodiment C37.2, wherein the one or moremyosin II inhibitors comprise one or more small molecule myosin IIinhibitors.

C37.4 The epithelial cells of embodiment C37.2 or C37.3, wherein the oneor more myosin II inhibitors comprise blebbistatin.

C38. The epithelial cells of any one of embodiments C1 to C37.4, whereinthe method further comprises increasing intracellular cyclic adenosinemonophosphate (cAMP) levels in the epithelial cells during the culturingin (a).

C39. The epithelial cells of embodiment C38, wherein increasingintracellular cyclic adenosine monophosphate (cAMP) levels comprises useof one or more beta-adrenergic agonists and/or one or morebeta-adrenergic receptor agonists.

C39.1 The epithelial cells of embodiment C39, where the one or morebeta-adrenergic agonists and/or the one or more beta-adrenergic receptoragonists comprise isoproterenol.

C40. The epithelial cells of any one of embodiments C1 to C40, whereinthe epithelial cells are obtained from a subject prior to (a).

C40.1 The epithelial cells of embodiment C40, wherein the subject is amammal.

C40.2 The epithelial cells of embodiment C40, wherein the subject is ahuman.

C40.3 The epithelial cells of any one of embodiments C40 to C40.2,wherein the epithelial cells are from tissue from a subject.

C40.4 The epithelial cells of embodiment C40.3, wherein the epithelialcells are from differentiated tissue from a subject.

C40.5 The epithelial cells of any one of embodiments C40 to C40.2,wherein the epithelial cells are from circulating cells from a subject.

C41. The epithelial cells of any one of embodiments C40 to C40.5,wherein the epithelial cells comprise primary cells from a subject.

C42. The epithelial cells of any one of embodiments C40 to C40.5,wherein the epithelial cells do not comprise primary cells from asubject.

C43. The epithelial cells of any one of embodiments C40 to C42, whereinthe epithelial cells comprise tumor cells from a subject.

C44. The epithelial cells of any one of embodiments C41 to C43, whereinthe epithelial cells from a subject are selected from squamous cells,columnar cells, adenomatous cells and transitional epithelial cells.

C44.1 The epithelial cells of any one of embodiments C41 to C43, whereinthe epithelial cells from a subject comprise one or more of squamouscells, columnar cells, adenomatous cells and transitional epithelialcells.

C45. The epithelial cells of any one of embodiments C41 to C44.1,wherein the epithelial cells from a subject comprise keratinocyteepithelial cells.

C45.1 The epithelial cells of embodiment C45, wherein the keratinocyteepithelial cells are selected from dermal keratinocyte, ocularepithelial cells, corneal epithelial cells, oral mucosal epithelialcells, esophagus epithelial cells, and cervix epithelial cells.

C46. The epithelial cells of any one of embodiments C41 to C44, whereinthe epithelial cells from a subject comprise non-keratinocyte epithelialcells.

C47. The epithelial cells of embodiment C46, wherein thenon-keratinocyte epithelial cells comprise glandular epithelial cells.

C48. The epithelial cells of embodiment C46 or C47, wherein thenon-keratinocyte epithelial cells are selected from prostate cells,mammary cells, hepatocytes, liver epithelial cells, biliary epithelialcells, gall bladder cells, pancreatic islet cells, pancreatic betacells, pancreatic ductal epithelial cells, pulmonary epithelial cells,airway epithelial cells, nasal epithelial cells, kidney cells, bladdercells, urethral epithelial cells, stomach epithelial cells, largeintestinal epithelial cells, small intestinal epithelial cells,testicular epithelial cells, ovarian epithelial cells, fallopian tubeepithelial cells, thyroid cells, parathyroid cells, adrenal cells,thymus cells, pituitary cells, glandular cells, amniotic epithelialcells, retinal pigmented epithelial cells, sweat gland epithelial cells,sebaceous epithelial cells and hair follicle cells.

C48.1 The epithelial cells of any one of embodiments C1 to C48, whereinthe epithelial cells comprise basal epithelial cells.

C48.2 The epithelial cells of any one of embodiments C1 to C48, whereinthe epithelial cells are not intestinal epithelial cells.

C49. The epithelial cells of any one of embodiments C1 to C48.2, whereinthe culturing in (a) is performed in the presence of a serum-freemedium.

C49.1 The epithelial cells of embodiment C49, wherein the serum-freemedium is a defined serum-free medium.

C49.2 The epithelial cells of embodiment C49, wherein the serum-freemedium is a xeno-free serum-free medium.

C49.3 The epithelial cells of embodiment C49, wherein the serum-freemedium is a defined xeno-free serum-free medium.

C50. The epithelial cells of any one of embodiments C49 to C49.3,wherein the serum-free medium comprises calcium.

C51. The epithelial cells of embodiment C50, wherein the serum-freemedium comprises calcium at a concentration below 1 mM.

C52. The epithelial cells of embodiment C50, wherein the serum-freemedium comprises calcium at a concentration below 500 μM.

C53. The epithelial cells of embodiment C50, wherein the serum-freemedium comprises calcium at a concentration below 100 μM.

C53.1 The epithelial cells of embodiment C53, wherein the serum-freemedium comprises calcium at a concentration of about 90 μM.

C54. The epithelial cells of embodiment C50, wherein the serum-freemedium comprises calcium at a concentration below 20 μM.

C54.1 The epithelial cells of any one of embodiments C49 to C54, whereinthe serum-free medium comprises a buffer and one or more of inorganicacids, salts, alkali silicates, amino acids, vitamins, purines,pyrimidines, polyamines, alpha-keto acids, organosulphur compounds andglucose.

C54.2 The epithelial cells of embodiment C54.1, wherein the one or moresalts are selected from sodium chloride, potassium chloride, sodiumacetate, and sodium phosphate.

C54.3 The epithelial cells of embodiment C54.1 or C54.2, wherein the oneor more amino acids are selected from arginine and glutamine.

C54.4 The epithelial cells of any one of embodiments C54.1 to C54.3,wherein the buffer is HEPES buffer.

C54.5 The epithelial cells any one of embodiments C49 to C54.4, whereinthe serum-free medium comprises albumin.

C54.6 The epithelial cells of embodiment C54.5, wherein the albumin isselected from bovine serum albumin and recombinant human serum albumin.

C54.7 The epithelial cells any one of embodiments C49 to C54.6, whereinthe serum-free medium comprises one or more lipids.

C54.8 The epithelial cells of embodiment C54.7, wherein the one or morelipids are selected from arachidonic acid, cholesterol,DL-alpha-tocopherol acetate, linoleic acid, linolenic acid, myristicacid, oleic acid, palmitic acid, palmitoleic acid, pluronic F-68,stearic acid, and polysorbate 80.

C54.9 The epithelial cells of embodiment C54.8, wherein the one or morelipids are selected from linoleic acid, linolenic acid, oleic acid,palmitic acid, and stearic acid.

C55. The epithelial cells of any one of embodiments C1 to C54, whereinthe method comprises use of one or more mitogenic growth factors.

C56. The epithelial cells of embodiment C55, wherein the one or moremitogenic growth factors comprise EGF.

C56.1 The epithelial cells of embodiment C55, wherein the one or moremitogenic growth factors comprise FGF.

C56.2 The epithelial cells of embodiment C55, wherein the one or moremitogenic growth factors comprise EGF and FGF.

C56.3 The epithelial cells of embodiment C56.1 or C56.2, wherein the FGFcomprises acidic FGF.

C57. The epithelial cells of any one of embodiments C1 to C54.9, whereinthe method does not comprise use of a mitogenic growth factor.

C58. The epithelial cells of any one of embodiments C1 to C57, whereinthe method comprises use of one or more mitogenic supplements.

C59. The epithelial cells of embodiment C58, wherein the one or moremitogenic supplements comprise bovine pituitary extract (BPE).

C59.1 The epithelial cells of any one of embodiments C1 to C57, whereinthe method does not comprise use of a mitogenic supplement.

C60. The epithelial cells of any one of embodiments C1 to C59.1, whereinthe method does not comprise use of a Wnt agonist or a beta-cateninagonist.

C60.1 The epithelial cells of any one of embodiments C1 to C60, whereinthe method does not comprise use of one or more of components selectedfrom: noggin, R-spondin, Wnt-3a, EGF, nicotinamide, FGF10, gastrin, ap38 inhibitor, SB202190, DHT, a notch inhibitor, a gamma secretaseinhibitor, DBZ and DAPT.

C61. The epithelial cells of any one of embodiments C1 to C60.1, whereinthe method does not comprise use of an extracellular matrix.

C62. The epithelial cells of any one of embodiments C1 to C60.1, whereinthe culturing in (a) is performed in a container comprising a coating.

C63. The epithelial cells of embodiment C62, wherein the coatingcomprises collagen.

C64. The epithelial cells of embodiment C62, wherein the coatingcomprises a basement membrane matrix.

C65. The epithelial cells of any one of embodiments C1 to C64, whereinthe culturing in (a) comprises expanding the epithelial cells.

C66. The epithelial cells of embodiment C65, wherein the epithelialcells are expanded at least about 2-fold.

C67. The epithelial cells of embodiment C65, wherein the epithelialcells are expanded at least about 5-fold.

C68. The epithelial cells of embodiment C65, wherein the epithelialcells are expanded at least about 10-fold.

C69. The epithelial cells of embodiment C65, wherein the epithelialcells are expanded at least about 15-fold.

C70. The epithelial cells of embodiment C65, wherein the epithelialcells are expanded at least about 20-fold.

C70.1 The epithelial cells of embodiment A65, wherein the epithelialcells are expanded at least about 100-fold.

C70.2 The epithelial cells of embodiment A65, wherein the epithelialcells are expanded at least about 1,000-fold.

C70.3 The epithelial cells of embodiment A65, wherein the epithelialcells are expanded at least about 10,000-fold.

C70.4 The epithelial cells of embodiment A65, wherein the epithelialcells are expanded at least about 100,000-fold.

C70.5 The epithelial cells of embodiment A65, wherein the epithelialcells are expanded at least about 1 million-fold.

C70.6 The epithelial cells of embodiment A65, wherein the epithelialcells are expanded at least about 1 billion-fold.

C70.7 The epithelial cells of embodiment A65, wherein the epithelialcells are expanded at least about 1 trillion-fold.

C71. The epithelial cells of any one of embodiments C65 to C70.7,wherein the epithelial cells are cultured for about 4 days.

C72. The epithelial cells of any one of embodiments C65 to C70.7,wherein the epithelial cells are cultured for about 5 days.

C73. The epithelial cells of any one of embodiments C1 to C72, whereinthe epithelial cells are continuously proliferated.

C74. The epithelial cells of any one of embodiments C1 to C73, whereinthe method comprises passaging the epithelial cells at least 15 times.

C75. The epithelial cells of any one of embodiments C1 to C73, whereinthe method comprises passaging the epithelial cells at least 25 times.

C76. The epithelial cells of any one of embodiments C1 to C75, wherein apopulation of the epithelial cells doubles over a period of time.

C77. The epithelial cells of embodiment C76, wherein the epithelial cellpopulation doubles at least 20 times.

C78. The epithelial cells of embodiment C76, wherein the epithelial cellpopulation doubles at least 50 times.

C78.1 The epithelial cells of embodiment C76, wherein the epithelialcell population doubles at least 80 times.

C79. The epithelial cells of embodiment C76, wherein the epithelial cellpopulation doubles at least 100 times.

C80. The epithelial cells of embodiment C76, wherein the epithelial cellpopulation doubles at least 120 times.

C81. The epithelial cells of embodiment C76, wherein the epithelial cellpopulation doubles at least 150 times.

C82. The epithelial cells of embodiment C76, wherein the epithelial cellpopulation doubles at least 200 times.

C83. The epithelial cells of any one of embodiments C76 to C82, whereinthe period of time is about 50 days.

C84. The epithelial cells of any one of embodiments C76 to C82, whereinthe period of time is about 100 days.

C85. The epithelial cells of any one of embodiments C76 to C82, whereinthe period of time is about 150 days.

C86. The epithelial cells of any one of embodiments C76 to C82, whereinthe period of time is about 200 days.

C87. The epithelial cells of any one of embodiments C1 to C86, whichcells maintain one or more native functional characteristics during (b).

C88. The epithelial cells of any one of embodiments C1 to C86, whichcells do not maintain one or more native functional characteristicsduring (b).

C89. The epithelial cells of any one of embodiments C1 to C88, whichcells are placed after (b) into a cell culture environment whereinTGF-beta signaling is not inhibited.

C90. The epithelial cells of embodiment C89, which cells maintain orregain one or more native functional characteristics after placementinto the cell culture environment wherein TGF-beta signaling is notinhibited.

C91. The epithelial cells of any one of embodiments C1 to C90, whichcells can be induced to differentiate into multiple tissue types.

C91.1 The epithelial cells of any one of embodiments C1 to C90, whichcells do not acquire the ability to differentiate into multiple tissuetypes.

C92. The epithelial cells of any one of embodiments C1 to C91.1, whichcells do not acquire the ability to form organoids.

C93. The epithelial cells of any one of embodiments C1 to C92, whichcells are not derived from embryonic stem cells.

C93.1 The epithelial cells of any one of embodiments C1 to C93, whichcells are not derived from continuously proliferating epithelial stemcells.

C93.2 The epithelial cells of any one of embodiments C1 to C93.1, whichcells are derived from epithelial tissue comprising quiescent epithelialcells.

C93.3 The epithelial cells of any one of embodiments C1 to C93.2, whichmethod does not comprise selecting for continuously proliferatingepithelial stem cells.

C93.4 The epithelial cells of any one of embodiments C1 to C93.3, whichmethod does not comprise selecting for intestinal crypt cells.

C93.5 The epithelial cells of any one of embodiments C1 to C93.4, whichmethod does not comprise selecting for LGR5+ cells.

C94. The epithelial cells of any one of embodiments C40 to C93.5,wherein the cells obtained from a subject and/or the population of exvivo proliferated cells do not comprise continuously proliferatingepithelial stem cells or cells derived from continuously proliferatingepithelial stem cells.

C94.1 The epithelial cells of any one of embodiments C40 to C94, whereinthe cells obtained from a subject and/or the population of ex vivoproliferated cells do not comprise pluripotent stem cells or cellsderived from pluripotent stem cells.

C94.2 The epithelial cells of any one of embodiments C40 to C94.1,wherein the cells obtained from a subject and/or the population of exvivo proliferated cells do not comprise terminally differentiatedepithelial cells.

C94.3 The epithelial cells of any one of embodiments C40 to C94.2,wherein the cells obtained from a subject and/or the population of exvivo proliferated cells do not comprise gastric epithelial cells,intestinal epithelial cells, and/or pancreatic epithelial cells.

C94.4 The epithelial cells of any one of embodiments C40 to C94.3,wherein the cells obtained from a subject and/or the population of exvivo proliferated cells do not comprise intestinal crypt cells.

C94.5 The epithelial cells of any one of embodiments C40 to C94.4,wherein the cells obtained from a subject and/or the population of exvivo proliferated cells do not comprise LGR5+ cells.

C95. The epithelial cells of any one of embodiments C40 to C94.5,wherein the cells obtained from a subject and/or the population of exvivo proliferated cells are a homogenous population of epithelial cells.

C95.1 The epithelial cells of any one of embodiments C40 to C95, whereinthe cells obtained from a subject and/or the population of ex vivoproliferated cells are a homogenous population of basal epithelialcells.

C95.2 The epithelial cells of any one of embodiments C40 to C94.5,wherein the cells obtained from a subject and/or the population of exvivo proliferated cells are a heterogeneous population of epithelialcells.

C96. The epithelial cells of any one of embodiments C40 to C95.2,wherein the cells obtained from a subject and/or the population of exvivo proliferated cells are less differentiated than terminallydifferentiated cells and are more differentiated than embryonic stemcells or adult stem cells.

C97. The epithelial cells of any one of embodiments C40 to C96, whereinthe cells obtained from a subject and/or the population of ex vivoproliferated cells express one or more basal epithelial cell markers.

C98. The epithelial cells of any one of embodiments C40 to C97, whereinthe cells obtained from a subject and/or the population of ex vivoproliferated cells express one or more of ITGA6, ITGB4, KRT14, KRT15,KRT5 and TP63.

C99. The epithelial cells of any one of embodiments C40 to C98, whereinthe cells obtained from a subject and/or the population of ex vivoproliferated cells do not express one or more epithelial stem cellmarkers.

C100. The epithelial cells of any one of embodiments C40 to C99, whereinthe cells obtained from a subject and/or the population of ex vivoproliferated cells do not express Lgr5.

C101. The epithelial cells of any one of embodiments C40 to C100,wherein the cells obtained from a subject and/or the population of exvivo proliferated cells do not express one or more pluripotent stem cellmarkers.

C102. The epithelial cells of any one of embodiments C40 to C101,wherein the cells obtained from a subject and/or the population of exvivo proliferated cells do not express one or more of LIN28A, NANOG,POU5F1/OCT4 and SOX2.

C103. The epithelial cells of any one of embodiments C40 to C102,wherein the cells obtained from a subject and/or the population of exvivo proliferated cells do not express one or more terminallydifferentiated epithelial cell markers.

C104. The epithelial cells of any one of embodiments C40 to C103,wherein the cells obtained from a subject and/or the population of exvivo proliferated cells do not express one or more of CFTR, FOXJ1, IVL,KRT1, KRT10, KRT20, LOR, MUC1, MUC5AC, SCGB1A1, SFTPB and SFTPD.

C105. The epithelial cells of any one of embodiments C40 to C104,wherein the cells obtained from a subject and/or the population of exvivo proliferated cells do not express one or more gastric epithelialcell markers, one or more intestinal epithelial cell markers, and/or oneor more pancreatic epithelial cell markers.

C106. The epithelial cells of any one of embodiments C40 to C105,wherein the cells obtained from a subject and/or the population of exvivo proliferated cells do not express one or more of CD34, HNF1A,HNF4A, IHH, KIT, LGR5, PDX1, and PROM1/CD133.

C107. Use of the epithelial cells of any one of embodiments C1 to C106for production of genetically modified cells.

C108. Use of the epithelial cells of any one of embodiments C1 to C106for identifying one or more candidate treatments for a subject.

C109. Use of the epithelial cells of any one of embodiments C1 to C106for identifying one or more abnormal epithelial cells in a subject.

C110. Use of the epithelial cells of any one of embodiments C1 to C106for monitoring the progression of a disease or treatment of a disease ina subject.

D1. A cell culture composition comprising a defined serum-free cellculture medium, a lipids mix, EGF, FGF, albumin, a TGF-beta inhibitor ora TGF-beta signaling inhibitor, and a Rho kinase inhibitor or aRho-associated protein kinase inhibitor.

D2. A cell culture composition consisting of a defined serum-free cellculture medium, a lipids mix, EGF, FGF, albumin, a TGF-beta inhibitor ora TGF-beta signaling inhibitor, and a Rho kinase inhibitor or aRho-associated protein kinase inhibitor.

D3. A cell culture composition comprising a defined serum-free cellculture medium, a lipids mix, EGF, FGF, albumin, a TGF-beta inhibitor ora TGF-beta signaling inhibitor, a Rho kinase inhibitor or aRho-associated protein kinase inhibitor, and a beta-adrenergic agonistor a beta-adrenergic receptor agonist.

D4. A cell culture composition consisting of a defined serum-free cellculture medium, a lipids mix, EGF, FGF, albumin, a TGF-beta inhibitor ora TGF-beta signaling inhibitor, a Rho kinase inhibitor or aRho-associated protein kinase inhibitor, and a beta-adrenergic agonistor a beta-adrenergic receptor agonist.

D5. A cell culture composition comprising a xeno-free serum-free cellculture medium, a lipids mix, EGF, FGF, albumin, a TGF-beta inhibitor ora TGF-beta signaling inhibitor, and a Rho kinase inhibitor or aRho-associated protein kinase inhibitor.

D6. A cell culture composition consisting of a xeno-free serum-free cellculture medium, a lipids mix, EGF, FGF, albumin, a TGF-beta inhibitor ora TGF-beta signaling inhibitor, and a Rho kinase inhibitor or aRho-associated protein kinase inhibitor.

D7. A cell culture composition comprising a xeno-free serum-free cellculture medium, a lipids mix, EGF, FGF, albumin, a TGF-beta inhibitor ora TGF-beta signaling inhibitor, a Rho kinase inhibitor or aRho-associated protein kinase inhibitor, and a beta-adrenergic agonistor a beta-adrenergic receptor agonist.

D8. A cell culture composition consisting of a xeno-free serum-free cellculture medium, a lipids mix, EGF, FGF, albumin, a TGF-beta inhibitor ora TGF-beta signaling inhibitor, a Rho kinase inhibitor or aRho-associated protein kinase inhibitor, and a beta-adrenergic agonistor a beta-adrenergic receptor agonist.

D9. A cell culture composition comprising a defined xeno-free serum-freecell culture medium, a lipids mix, EGF, FGF, albumin, a TGF-betainhibitor or a TGF-beta signaling inhibitor, and a Rho kinase inhibitoror a Rho-associated protein kinase inhibitor.

D10. A cell culture composition consisting of a defined xeno-freeserum-free cell culture medium, a lipids mix, EGF, FGF, albumin, aTGF-beta inhibitor or a TGF-beta signaling inhibitor, and a Rho kinaseinhibitor or a Rho-associated protein kinase inhibitor.

D11. A cell culture composition comprising a defined xeno-freeserum-free cell culture medium, a lipids mix, EGF, FGF, albumin, aTGF-beta inhibitor or a TGF-beta signaling inhibitor, a Rho kinaseinhibitor or a Rho-associated protein kinase inhibitor, and abeta-adrenergic agonist or a beta-adrenergic receptor agonist.

D12. A cell culture composition consisting of a defined xeno-freeserum-free cell culture medium, a lipids mix, EGF, FGF, albumin, aTGF-beta inhibitor or a TGF-beta signaling inhibitor, a Rho kinaseinhibitor or a Rho-associated protein kinase inhibitor, and abeta-adrenergic agonist or a beta-adrenergic receptor agonist.

E1. A method for proliferating epithelial cells ex vivo, comprising:

-   -   expanding the number of cells in an originating epithelial cell        population derived from differentiated tissue under feeder-cell        free expansion culture conditions, thereby generating an        expanded epithelial cell population, wherein:    -   the expansion culture conditions comprise an agent that        activates telomerase reverse transcriptase in the population        and/or inhibits transforming growth factor beta (TGF-beta)        signaling in the population;    -   the originating epithelial cell population is capable of 25        population doublings or more when cultured under the expansion        culture conditions; and    -   the originating epithelial cell population is capable of no more        than 20 population doublings when cultured under control culture        conditions that do not include the agent.

E1.1 A method for proliferating epithelial cells ex vivo, comprising:

-   -   expanding the number of cells in an originating epithelial cell        population derived from differentiated tissue under serum-free        and feeder-cell free conditions, thereby generating an expanded        epithelial cell population, wherein:    -   the expansion culture conditions comprise an agent that        activates telomerase reverse transcriptase in the population        and/or inhibits transforming growth factor beta (TGF-beta)        signaling in the population; and    -   the originating epithelial cell population comprises quiescent        and/or formerly quiescent epithelial cells.

E1.2 The method of embodiment E1, wherein:

-   -   agent that activates telomerase reverse transcriptase in the        population and/or inhibits transforming growth factor beta        (TGF-beta) signaling in the population is a first agent,    -   the expansion culture conditions further comprise a second agent        that modulates cytoskeletal structure in the population, and    -   the control culture conditions do not include the first agent        and the second agent.

E1.3 The method of embodiment E1.1, wherein the expansion cultureconditions further comprise an agent that modulates cytoskeletalstructure in the population.

E2. The method of any one of embodiments E1 to E1.3, comprisingisolating the originating epithelial cell population from thedifferentiated tissue.

E3. The method of any one of embodiments E1 to E2, comprisingmaintaining or proliferating cells of the originating epithelial cellpopulation in cell culture after the cells are isolated from thedifferentiated tissue and prior to contacting the originating epithelialcell population to the feeder-cell free expansion culture conditions.

E4. The method of any one of embodiments E1 to E3, wherein theoriginating epithelial cell population and the expanded epithelial cellpopulation contain no embryonic stem cells.

E5. The method of any one of embodiments E1 to E4, wherein:

-   -   the originating epithelial cell population, or    -   the expanded epithelial cell population, or    -   the originating epithelial cell population and the expanded        epithelial cell population,    -   express one or more basal epithelial cell markers.

E6. The method of any one of embodiments E1 to E5, wherein:

-   -   the originating epithelial cell population, or    -   the expanded epithelial cell population, or    -   the originating epithelial cell population and the expanded        epithelial cell population,    -   express one or more of ITGA6, ITGB4, KRT14, KRT15, KRT5 and        TP63.

E7. The method of any one of embodiments E1 to E6, wherein:

-   -   the originating epithelial cell population, or    -   the expanded epithelial cell population, or    -   the originating epithelial cell population and the expanded        epithelial cell population,    -   do not express one or more epithelial stem cell markers.

E8. The method of any one of embodiments E1 to E7, wherein:

-   -   the originating epithelial cell population, or    -   the expanded epithelial cell population, or    -   the originating epithelial cell population and the expanded        epithelial cell population,    -   do not express Lgr5.

E9. The method of any one of embodiments E1 to E8, wherein:

-   -   the originating epithelial cell population, or    -   the expanded epithelial cell population, or    -   the originating epithelial cell population and the expanded        epithelial cell population,    -   do not express one or more pluripotent stem cell markers.

E10. The method of any one of embodiments E1 to E9, wherein:

-   -   the originating epithelial cell population, or    -   the expanded epithelial cell population, or    -   the originating epithelial cell population and the expanded        epithelial cell population,    -   do not express one or more of LIN28A, NANOG, POU5F1/OCT4 and        SOX2.

E11. The method of any one of embodiments E1 to E10, wherein:

-   -   the originating epithelial cell population, or    -   the expanded epithelial cell population, or    -   the originating epithelial cell population and the expanded        epithelial cell population,    -   do not express one or more terminally differentiated epithelial        cell markers.

E12. The method of any one of embodiments E1 to E11, wherein:

-   -   the originating epithelial cell population, or    -   the expanded epithelial cell population, or    -   the originating epithelial cell population and the expanded        epithelial cell population,    -   do not express one or more of CFTR, FOXJ1, IVL, KRT1, KRT10,        KRT20, LOR, MUC1, MUC5AC, SCGB1A1, SFTPB and SFTPD.

E13. The method of any one of embodiments E1 to E12, wherein:

-   -   the originating epithelial cell population, or    -   the expanded epithelial cell population, or    -   the originating epithelial cell population and the expanded        epithelial cell population,    -   do not express one or more gastric epithelial cell markers, one        or more intestinal epithelial cell markers, and/or one or more        pancreatic epithelial cell markers.

E14. The method of any one of embodiments E1 to E13, wherein:

-   -   the originating epithelial cell population, or    -   the expanded epithelial cell population, or    -   the originating epithelial cell population and the expanded        epithelial cell population,    -   do not express one or more of CD34, HNF1A, HNF4A, IHH, KIT,        LGR5, PDX1, and PROM1/CD133.

E15. The method of any one of embodiments E1 to E14, wherein:

-   -   the originating epithelial cell population, or    -   the expanded epithelial cell population, or    -   the originating epithelial cell population and the expanded        epithelial cell population,    -   comprise quiescent and/or formerly quiescent epithelial cells.

E16. The method of any one of embodiments E1 to E15, wherein the agentthat activates telomerase reverse transcriptase in the population and/orinhibits transforming growth factor beta (TGF-beta) signaling comprisesone or more TGF-beta signaling inhibitors.

E17. The method of embodiment E16, wherein the one or more TGF-betasignaling inhibitors comprise one or more ALK5 inhibitors.

E18. The method of embodiment E17, wherein the one or more ALK5inhibitors comprise one or more small molecule ALK5 inhibitors.

E19. The method of embodiment E17, wherein the one or more ALK5inhibitors are selected from A83-01, GW788388, RepSox, and SB 431542.

E20. The method of any one of embodiments E1 to E19, wherein the agentthat modulates cytoskeletal structure comprises one or more of aRho-associated protein kinase inhibitor, a p21-activated kinase (PAK)inhibitor, and a myosin II inhibitor.

E21. The method of embodiment E20, wherein the Rho kinase inhibitor isselected from a Rho-associated protein kinase 1 (ROCK 1) inhibitor and aRho-associated protein kinase 2 (ROCK 2) inhibitor.

E22. The method of embodiment E20 or E21, wherein the Rho-associatedprotein kinase inhibitor comprises one or more small moleculeRho-associated protein kinase inhibitors.

E23. The method of embodiment E22, wherein the one or moreRho-associated protein kinase inhibitors is selected from Y-27632, SR3677, thiazovivin, HA1100 hydrochloride, HA1077 and GSK-429286.

E24. The method of any one of embodiments E20 to E23, wherein thep21-activated kinase (PAK) inhibitor is selected from a PAK1 inhibitor,a PAK2 inhibitor, a PAK3 inhibitor and a PAK4 inhibitor.

E25. The method of embodiment E24, wherein the p21-activated kinase(PAK) inhibitor comprises one or more small molecule PAK1 inhibitors.

E26. The method of embodiment E25, wherein the one or more smallmolecule PAK1 inhibitors comprise IPA3.

E27. The method of any one of embodiments E20 to E26, wherein the myosinII inhibitor comprises one or more non-muscle myosin II (NM II)inhibitors.

E28. The method of embodiment E27, wherein the one or more non-musclemyosin II (NM II) inhibitors comprise one or more small moleculenon-muscle myosin II (NM II) inhibitors.

E29. The method of embodiment E28, wherein the one or more smallmolecule non-muscle myosin II (NM II) inhibitors comprise blebbistatin.

E30. The method of any one of embodiments E1 to E29, wherein theexpansion culture conditions further comprise an agent that increasesintracellular cyclic adenosine monophosphate (cAMP) levels in thepopulation.

E31. The method of embodiment E30, wherein the agent that increasesintracellular cyclic adenosine monophosphate (cAMP) levels comprises oneor more beta-adrenergic receptor agonists.

E32. The method of embodiment E31, wherein the one or morebeta-adrenergic receptor agonists comprise isoproterenol.

E33. The method of any one of embodiments E1 to E32, wherein theexpansion culture conditions are serum-free culture conditions.

E34. The method of any one of embodiments E1 to E33, wherein theexpansion culture conditions are defined serum-free culture conditions.

E35. The method of any one of embodiments E1 to E34, wherein theexpansion culture conditions are xeno-free culture conditions.

E36. The method of any one of embodiments E1 to E35, wherein theexpansion culture conditions are defined xeno-free culture conditions.

E37. The method of any one of embodiments E1 to E36, wherein theexpansion culture conditions comprise calcium at a concentration below100 μM.

E38. The method of embodiment E37, wherein the calcium is present at aconcentration of about 90 μM.

E39. The method of any one of embodiments E1 to E38, wherein theexpansion culture conditions comprise one or more mitogenic growthfactors.

E40. The method of embodiment E39, wherein one or more mitogenic growthfactors comprise EGF, FGF, or EGF and FGF.

E41. The method of any one of embodiments E1 to E40, wherein theexpansion culture conditions comprise no extracellular matrix.

E42. The method of any one of embodiments E1 to E41, wherein theoriginating epithelial cell population is capable of 30 populationdoublings or more when cultured under the expansion culture conditions.

E43. The method of any one of embodiments E1 to E41, wherein theoriginating epithelial cell population is capable of 50 populationdoublings or more when cultured under the expansion culture conditions.

E44. The method of any one of embodiments E1 to E41, wherein theoriginating epithelial cell population is capable of 80 populationdoublings or more when cultured under the expansion culture conditions.

E45. The method of any one of embodiments E1 to E41, wherein theoriginating epithelial cell population is capable of 100 populationdoublings or more when cultured under the expansion culture conditions.

E46. The method of any one of embodiments E1 to E45, wherein the methoddoes not comprise selecting for continuously proliferating epithelialstem cells in the originating epithelial cell population.

E47. The method of any one of embodiments E1 to E45, wherein theoriginating epithelial cell population does not comprise continuouslyproliferating epithelial stem cells.

E48. The method of any one of embodiments E1 to E47, further comprisingisolating a population of ex vivo expanded epithelial cells.

E49. The method of any one of embodiments E1 to E48, further comprisingstoring a population of ex vivo expanded epithelial cells in a cellbank.

E50. A population of ex vivo expanded epithelial cells produced by amethod according to any one of embodiments E1 to E49.

E51. Use of the population of ex vivo expanded epithelial cells ofembodiment E50 for production of genetically modified cells.

E52. Use of the population of ex vivo expanded epithelial cells ofembodiment E50 for identifying one or more candidate treatments for asubject.

E53. Use of the population of ex vivo expanded epithelial cells ofembodiment E50 for identifying one or more abnormal epithelial cells ina subject.

E54. Use of the population of ex vivo expanded epithelial cells ofembodiment E50 for monitoring the progression of a disease or treatmentof a disease in a subject.

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents. Their citation is not an indication of asearch for relevant disclosures. All statements regarding the date(s) orcontents of the documents is based on available information and is notan admission as to their accuracy or correctness.

Modifications may be made to the foregoing without departing from thebasic aspects of the technology. Although the technology has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, yet these modifications and improvements are within thescope and spirit of the technology.

The technology illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising,” “consisting essentially of,” and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and use of such terms and expressions do not exclude anyequivalents of the features shown and described or portions thereof, andvarious modifications are possible within the scope of the technologyclaimed. The term “a” or “an” can refer to one of or a plurality of theelements it modifies (e.g., “a reagent” can mean one or more reagents)unless it is contextually clear either one of the elements or more thanone of the elements is described. The term “about” as used herein refersto a value within 10% of the underlying parameter (i.e., plus or minus10%), and use of the term “about” at the beginning of a string of valuesmodifies each of the values (i.e., “about 1, 2 and 3” refers to about 1,about 2 and about 3). For example, a weight of “about 100 grams” caninclude weights between 90 grams and 110 grams. Further, when a listingof values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or86%) the listing includes all intermediate and fractional values thereof(e.g., 54%, 85.4%). Thus, it should be understood that although thepresent technology has been specifically disclosed by representativeembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and such modifications and variations are considered within thescope of this technology.

Certain embodiments of the technology are set forth in the claim(s) thatfollow(s).

What is claimed is:
 1. A method for proliferating epithelial cells exvivo, comprising: expanding the number of cells in an originatingepithelial cell population derived from differentiated tissue, embryonicstem (ES) cells, or induced pluripotent stem cells (iPSCs) underexpansion culture conditions, thereby generating an expanded epithelialcell population, wherein: the expansion culture conditions comprise afirst agent consisting of an ALK5 inhibitor and a second agent chosenfrom a Rho-associated protein kinase inhibitor, a p21-activated kinase(PAK) inhibitor, and a myosin II inhibitor; the expansion cultureconditions are serum-free and feeder-cell free culture conditions, andare defined culture conditions, xeno-free culture conditions, or definedand xeno-free culture conditions; the originating epithelial cellpopulation is capable of 25 population doublings or more when culturedunder the expansion culture conditions; and the originating epithelialcell population is capable of no more than 20 population doublings whencultured under control culture conditions that do not include the firstagent and the second agent.
 2. The method of claim 1, wherein the ALK5inhibitor is selected from A83-01, GW788388, RepSox, and SB
 431542. 3.The method of claim 1, wherein the second agent is a Rho-associatedprotein kinase inhibitor.
 4. The method of claim 3, wherein theRho-associated protein kinase inhibitor is selected from Y-27632, SR3677, thiazovivin, HA1100 hydrochloride, HA1077 and GSK-429286.
 5. Themethod of claim 1, wherein the second agent is a PAK inhibitor.
 6. Themethod of claim 5, wherein the PAK inhibitor is IPA3.
 7. The method ofclaim 1, wherein the second agent is a myosin II inhibitor.
 8. Themethod of claim 7, wherein the myosin II inhibitor is blebbistatin. 9.The method of claim 1, wherein the expansion culture conditions comprisea beta-adrenergic receptor agonist.
 10. The method of claim 9, whereinthe beta-adrenergic receptor agonist is isoproterenol.
 11. The method ofclaim 1, wherein the expansion culture conditions comprise calcium at aconcentration below 200 μM.
 12. The method of claim 1, wherein theexpansion culture conditions comprise calcium at a concentration below100 μM.
 13. The method of claim 1, wherein the expansion cultureconditions comprise one or more mitogenic growth factors.
 14. The methodof claim 13, wherein the one or more mitogenic growth factors compriseEGF, FGF, or EGF and FGF.
 15. The method of claim 1, wherein theoriginating epithelial cell population is capable of 50 populationdoublings or more when cultured under the expansion culture conditions.16. The method of claim 1, wherein the originating epithelial cellpopulation is capable of 80 population doublings or more when culturedunder the expansion culture conditions.
 17. The method of claim 1,wherein the originating epithelial cell population is capable of 100population doublings or more when cultured under the expansion cultureconditions.
 18. The method of claim 1, wherein the expansion cultureconditions do not comprise one or more of extracellular matrixcomprising heterogeneous components, a Wnt agonist or a beta-cateninagonist.
 19. The method of claim 1, wherein the expansion cultureconditions comprise inhibitors consisting of an ALK5 inhibitor and aRho-associated protein kinase inhibitor.
 20. The method of claim 19,wherein the expansion culture conditions comprise inhibitors consistingof A83-01 and Y-27632.
 21. The method of claim 1, wherein the expansionculture conditions comprise inhibitors consisting of an ALK5 inhibitorand a p21-activated kinase (PAK) inhibitor.
 22. The method of claim 21,wherein the expansion culture conditions comprise inhibitors consistingof A83-01 and IPA3.
 23. The method of claim 1, wherein the expansionculture conditions comprise inhibitors consisting of an ALK5 inhibitorand a myosin II inhibitor.
 24. The method of claim 23, wherein theexpansion culture conditions comprise inhibitors consisting of A83-01and blebbistatin.
 25. A method for proliferating epithelial cells exvivo, comprising: expanding the number of cells in an originatingepithelial cell population derived from differentiated tissue, embryonicstem (ES) cells, or induced pluripotent stem cells (iPSCs) underexpansion culture conditions, thereby generating an expanded epithelialcell population, wherein: the expansion culture conditions comprisecomponents consisting essentially of i) a transforming growth factorbeta (TGF-beta) signaling inhibitor; ii) a Rho-associated protein kinaseinhibitor, a p21-activated kinase (PAK) inhibitor, or a myosin IIinhibitor; iii) a mitogenic growth factor; iv) a beta-adrenergicreceptor agonist; v) calcium at a concentration below 100 μM; and vi) aserum-free base medium; the expansion culture conditions are serum-freeand feeder-cell free culture conditions, and are defined cultureconditions, xeno-free culture conditions, or defined and xeno-freeculture conditions; the originating epithelial cell population iscapable of 25 population doublings or more when cultured under theexpansion culture conditions; and the originating epithelial cellpopulation is capable of no more than 20 population doublings whencultured under control culture conditions that do not include the firstagent and the second agent.
 26. A population of ex vivo expandedepithelial cells produced by a method comprising: expanding the numberof cells in an originating epithelial cell population derived fromdifferentiated tissue, embryonic stem (ES) cells, or induced pluripotentstem cells (iPSCs) under expansion culture conditions, therebygenerating an expanded epithelial cell population, wherein: theexpansion culture conditions comprise a first agent consisting of anALK5 inhibitor and a second agent chosen from a Rho-associated proteinkinase inhibitor, a p21-activated kinase (PAK) inhibitor, and a myosinII inhibitor; the expansion culture conditions are serum-free andfeeder-cell free culture conditions, and are defined culture conditions,xeno-free culture conditions, or defined and xeno-free cultureconditions; the originating epithelial cell population is capable of 25population doublings or more when cultured under the expansion cultureconditions; and the originating epithelial cell population is capable ofno more than 20 population doublings when cultured under control cultureconditions that do not include the first agent and the second agent. 27.A method for genetically modifying cells, comprising contacting cellswith one or more agents that introduce a genetic modification to thecells, wherein the cells have been expanded by a method comprising:expanding the number of cells in an originating epithelial cellpopulation derived from differentiated tissue, embryonic stem (ES)cells, or induced pluripotent stem cells (iPSCs) under expansion cultureconditions, thereby generating an expanded epithelial cell population,wherein: the expansion culture conditions comprise a first agentconsisting of an ALK5 inhibitor and a second agent chosen from aRho-associated protein kinase inhibitor, a p21-activated kinase (PAK)inhibitor, and a myosin II inhibitor; the expansion culture conditionsare serum-free and feeder-cell free culture conditions, and are definedculture conditions, xeno-free culture conditions, or defined andxeno-free culture conditions; the originating epithelial cell populationis capable of 25 population doublings or more when cultured under theexpansion culture conditions; and the originating epithelial cellpopulation is capable of no more than 20 population doublings whencultured under control culture conditions that do not include the firstagent and the second agent.